Meiotic arrest occurs most frequently at metaphase and is often incomplete in azoospermic men.

OBJECTIVE: To establish which meiotic checkpoints are activated in males with severe spermatogenic impairment to improve phenotypic characterization of meiotic defects. DESIGN: Retrospective observational study. SETTING: University medical center research laboratory and andrology clinic. PATIENT(S): Forty-eight patients with confirmed spermatogenic impairment (Johnsen scores 3-6) and 15 controls (Johnsen score 10). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Quantitative assessment of immunofluorescent analyses of specific markers to determine meiotic entry, chromosome pairing, progression of DNA double-strand break repair, crossover formation, formation of meiotic metaphases, metaphase arrest, and spermatid formation, resulting in a novel classification of human meiotic arrest types. RESULT(S): Complete metaphase arrest was observed most frequently (27%), and the patients with the highest frequency of apoptotic metaphases also displayed a reduction in crossover number. Incomplete metaphase arrest was observed in 17% of the patients. Only four patients (8%) displayed a failure to complete meiotic chromosome pairing leading to pachytene arrest. Two new types of meiotic arrest were defined: premetaphase and postmetaphase arrest (15% and 13%, respectively). CONCLUSION(S): Meiotic arrest in men occurs most frequently at meiotic metaphase. This arrest can be incomplete, resulting in low numbers of spermatids, and often occurs in association with reduced crossover frequency. The phenotyping approach described here provides mechanistic insights to help identify candidate infertility genes and to assess genotype-phenotype correlations in individual cases.

Novel Therapeutic Strategy for Pharyngoesophageal Stricture following Total Laryngectomy.

Current therapeutic strategies for pharyngoesophageal stricture, while effective in the short term, are protracted and costly in the longer term. Conceptually, if a stricture can be dilated with minimal tissue injuries, the rate of fibrosis and the resultant stricture recurrence could be reduced. We evaluated a prototype computer-controlled syringe pump device programmed to distend a commercially available balloon dilator at variable rate, asserting incremental lumen distension pressures tailored to the resistive force encountered within the stricture. We completed 17 graded dilatation procedures among 4 total laryngectomy patients. All patients had a short-term response (1 month), with a mean decrement (improvement) in Sydney Swallow Questionnaire score of 448 (total score range, 0-1700; normal <234). The overall procedural tolerability and safety were encouraging; the only complication was the displacement of the voice prosthesis during 1 dilatation. From a technical viewpoint, the main challenge was to maintain the balloon in position during dilatation.

Multiparameter Investigation of a 46,XX/46,XY Tetragametic Chimeric Phenotypical Male Patient with Bilateral Scrotal Ovotestes and Ovulatory Activity.

We report on an adult male initially presenting with gynecomastia and a painless scrotal mass without additional genital anomalies. Hyperpigmentation of the skin following the Blaschko's lines was identified. He underwent gonadectomy because of suspected cancer. Histological analyses revealed an ovotestis with ovulatory activity confirmed by immunohistochemistry with multiple markers. Karyotyping of cultured peripheral blood lymphocytes and a buccal smear revealed a 46,XX/46,XY chimeric constitution with different percentages. Multiple molecular analyses as well as blood typing implied a tetragametic origin. After the unilateral gonadectomy, the patient developed recurrent painful cystic swellings of the remaining gonad. Because of the wish to preserve hormonal activity as well as future fertility, the patient underwent surgical resection of a cystic gonadal area. The removed tissue showed ovulation-related features in addition to both testicular and ovarian tissue, diagnosed as an ovotestis. Testosterone therapy was initiated to suppress the persistently elevated gonadotropins and thereby suppress ovarian activity. During treatment, the recurrent pain complaints and cystic swellings ceased, although gonadotropin levels were not fully suppressed. Based on these observations, the importance of a detailed genetic and pathological diagnosis and the clinical dilemmas including the pros and cons of personalized treatment with gonadal preservative surgery are discussed.

A case of angioimmunoblastic T-cell non-Hodgkin lymphoma with a neocentric inv dup(1).

Neocentromeres are rare epigenetic phenomena in which functional centromeres are formed onto novel chromosomal locations without any alpha-satellite DNA. To date, constitutional human neocentromeres have been reported in at least 90 cases. In cancer, however, the knowledge is much more limited. Acquired neocentromeres have been described in a particular class of lipomatous tumors (atypical lipomas and well-differentiated liposarcomas; ALP-WDLPS), three cases of acute myeloid leukemia (AML), one case of non-Hodgkin lymphoma (NHL), and one case of lung carcinoma. Here, we report on a 66-year-old male with angioimmunoblastic T-cell NHL. Cytogenetic analysis of his bone marrow showed multiple aberrations, including the presence of a supernumerary chromosome. Using the fluorescence in situ hybridization technique, the supernumerary chromosome was demonstrated to be entirely composed of material derived from chromosome 1. It represented an inverted duplication of the segments between 1q21 and 1qter with a neocentromere in band 1q31. To our knowledge, this is the second reported case of NHL (both T-cell) with the presence of a neocentromere. The occurrence of neocentromeres in tumor cells, however, may be underestimated because of technical limitations during the routine diagnostic chromosomal analysis. The prognostic impact is therefore currently unknown.

Several tetratricopeptide repeat (TPR) motifs of FANCG are required for assembly of the BRCA2/D1-D2-G-X3 complex, FANCD2 monoubiquitylation and phleomycin resistance.

The Fanconi anaemia (FA) FANCG protein is an integral component of the FA nuclear core complex that is required for monoubiquitylation of FANCD2. FANCG is also part of another protein complex termed D1-D2-G-X3 that contains FANCD2 and the homologous recombination repair proteins BRCA2 (FANCD1) and XRCC3. Formation of the D1-D2-G-X3 complex is mediated by serine-7 phosphorylation of FANCG and occurs independently of the FA core complex and FANCD2 monoubiquitylation. FANCG contains seven tetratricopeptide repeat (TPR) motifs that mediate protein-protein interactions and here we show that mutation of several of the TPR motifs at a conserved consensus residue ablates the in vivo binding activity of FANCG. Expression of mutated TPR1, TPR2, TPR5 and TPR6 in Chinese hamster fancg mutant NM3 fails to functionally complement its hypersensitivities to mitomycin C (MMC) and phleomycin and fails to restore FANCD2 monoubiquitylation. Using co-immunoprecipitation analysis, we demonstrate that these TPR-mutated FANCG proteins fail to interact with BRCA2, XRCC3, FANCA or FANCF. The interactions of other proteins in the D1-D2-G-X3 complex are also absent, including the interaction of BRCA2 with both the monoubiquitylated (FANCD2-L) and non-ubiquitylated (FANCD2-S) isoforms of FANCD2. Interestingly, a mutation of TPR7 (R563E), that complements the MMC and phleomycin hypersensitivity of human FA-G EUFA316 cells, fails to complement NM3, despite the mutated FANCG protein co-precipitating with FANCA, BRCA2 and XRCC3. Whilst interaction of TPR7-mutated FANCG with FANCF does appear to be reduced in NM3, FANCD2 is monoubiquitylated suggesting that sub-optimal interactions of FANCG in the core complex and the D1-D2-G-X3 complex are responsible for the observed MMC- and phleomycin-hypersensitivity, rather than a defect in FANCD2 monoubiquitylation. Our data demonstrate that FANCG functions as a mediator of protein-protein interactions and is vital for the assembly of multi-protein complexes including the FA core complex and the D1-D2-G-X3 complex.

Tetratricopeptide-motif-mediated interaction of FANCG with recombination proteins XRCC3 and BRCA2.

Fanconi anaemia is an inherited chromosomal instability disorder characterised by cellular sensitivity to DNA interstrand crosslinkers, bone-marrow failure and a high risk of cancer. Eleven FA genes have been identified, one of which, FANCD1, is the breast cancer susceptibility gene BRCA2. At least eight FA proteins form a nuclear core complex required for monoubiquitination of FANCD2. The BRCA2/FANCD1 protein is connected to the FA pathway by interactions with the FANCG and FANCD2 proteins, both of which co-localise with the RAD51 recombinase, which is regulated by BRCA2. These connections raise the question of whether any of the FANC proteins of the core complex might also participate in other complexes involved in homologous recombination repair. We therefore tested known FA proteins for direct interaction with RAD51 and its paralogs XRCC2 and XRCC3. FANCG was found to interact with XRCC3, and this interaction was disrupted by the FA-G patient derived mutation L71P. FANCG was co-immunoprecipitated with both XRCC3 and BRCA2 from extracts of human and hamster cells. The FANCG-XRCC3 and FANCG-BRCA2 interactions did not require the presence of other FA proteins from the core complex, suggesting that FANCG also participates in a DNA repair complex that is downstream and independent of FANCD2 monoubiquitination. Additionally, XRCC3 and BRCA2 proteins co-precipitate in both human and hamster cells and this interaction requires FANCG. The FANCG protein contains multiple tetratricopeptide repeat motifs (TPRs), which function as scaffolds to mediate protein-protein interactions. Mutation of one or more of these motifs disrupted all of the known interactions of FANCG. We propose that FANCG, in addition to stabilising the FA core complex, may have a role in building multiprotein complexes that facilitate homologous recombination repair.

Spectrum and significance of variants and mutations in the Fanconi anaemia group G gene in children with sporadic acute myeloid leukaemia.

Childhood acute myeloid leukaemia (AML) is uncommon. Children with Fanconi anaemia (FA), however, have a very high risk of developing AML. FA is a rare inherited disease caused by mutations in at least 12 genes, of which Fanconi anaemia group G gene (FANCG) is one of the commonest. To address to what extent FANCG variants contribute to sporadic childhood AML, we determined the spectrum of FANCG sequence variants in 107 children diagnosed with sporadic AML, using polymerase chain reaction (PCR), fluorescent single-strand conformational polymorphism (SSCP) and sequencing methodologies. The significance of variants was determined by frequency analysis and assessment of evolutionary conservation. Seven children (6.5%) carried variants in FANCG. Two of these carried two variants, including the known IVS2 + 1G>A mutation with the novel missense mutation S588F, and R513Q with the intronic deletion IVS12-38 (-28)_del11, implying that these patients might have been undiagnosed FA patients. R513Q, which affects a semi-conserved amino acid, was carried in two additional children with AML. Although not significant, the frequency of R513Q was higher in children with AML than unselected cord bloods. While FANCG mutation carrier status does not predispose to sporadic AML, the identification of unrecognised FA patients implies that FA presenting with primary AML in childhood is more common than suspected.

Voice restoration with the advantage tracheoesophageal voice prosthesis.

OBJECTIVES: To determine whether the Blom-Singer indwelling Advantage tracheoesophageal voice prosthesis (TEP) extends prosthesis life span significantly in patients with documented premature device failure due to fungal colonization. STUDY DESIGN AND SETTING: Data were collected in a prospective manner on a total of 42 standard indwelling TEP users who exhibited early device failure, that is, between 2 weeks and 6 months, due to fungal colonization of the flap valve despite appropriate use of oral antifungal agents. There were 29 men and 13 women, whose ages ranged from 36 years 10 months to 86 years 8 months. METHODS: Baseline data were derived from the average number of days 3 previous standard indwelling prostheses functioned before leaking. An Advantage indwelling TEP was placed after the third change, oral antifungal agents stopped, and routine care implemented, that is, flush and brush the device in situ twice each day. Each participant was assigned to 1 of 3 groups. Group 1 had device failure equal to or less than 2 months (n = 12). Group 2 had device failure between 2 and 4 months (n = 19). Group 3 had device failure between 4 and 6 months (n = 11). RESULTS: Groups 1 and 2 exhibited significantly longer device life span, that is, 77 and 82 days, respectively (P < 0.01), and group 3 exhibited device life span that was longer but not significantly so, that is, 12 days (P > 0.05), after the change from standard to Advantage TEP. Individual data indicated that the majority of participants, that is, 32 of 42 (76.2%), experienced longer device life span after changing to the Advantage prosthesis. Specifically, 9 of 12 (75.0%) users in group 1, 17 of 19 (89.5%) users in group 2, and 6 of 11 (54.5%) users in group 3 exhibited longer device life span. The combination of using an Advantage TEP, discontinuing oral antifungal agents, and reducing the number of both TEP changes and clinic visits resulted in overall cost benefits for both the user and the health care system. The cost benefit for group 1 was dollar 520.00; group 2, dollar 393.00; and group 3, dollar 204.25. CONCLUSIONS: The Advantage TEP extended device life span significantly for standard indwelling device users with documented premature device failure due to fungal colonization, reduced costs associated with tracheoesophageal voice restoration rehabilitation, and enhanced user satisfaction by eliminating use of oral antifungal agents and reducing clinic visits. SIGNIFICANCE: Use of an Advantage indwelling voice prosthesis is warranted from both cost and user satisfaction perspectives when early and repeated device failure occurs as a result of fungal colonization. EBM RATING: B-3.

The Fanconi anemia gene product FANCF is a flexible adaptor protein.

The Fanconi anemia (FA) protein FANCF is an essential component of a nuclear core complex that protects the genome against chromosomal instability, but the specific function of FANCF is still poorly understood. Based upon the homology between human and Xenopus laevis FANCF, we carried out an extensive mutagenesis study to examine which domains are functionally important and to gain more insight into the function of FANCF. In contrast to previous suggestions, we show that FANCF does not have a ROM-like function. We found that the C terminus of FANCF interacts directly with FANCG and allows the assembly of other FA proteins into a stable complex. The N terminus appears to stabilize the interaction with FANCA and FANCG and is essential for the binding of the FANCC/FANCE subcomplex. We identified several important amino acids in this N-terminal region but, surprisingly, many amino acid changes failed to affect the function of the FANCF protein. Our data demonstrate that FANCF acts as a flexible adaptor protein that plays a key role in the proper assembly of the FA core complex.

Multiple TPR motifs characterize the Fanconi anemia FANCG protein.

The genome protection pathway that is defective in patients with Fanconi anemia (FA) is controlled by at least eight genes, including BRCA2. A key step in the pathway involves the monoubiquitylation of FANCD2, which critically depends on a multi-subunit nuclear 'core complex' of at least six FANC proteins (FANCA, -C, -E, -F, -G, and -L). Except for FANCL, which has WD40 repeats and a RING finger domain, no significant domain structure has so far been recognized in any of the core complex proteins. By using a homology search strategy comparing the human FANCG protein sequence with its ortholog sequences in Oryzias latipes (Japanese rice fish) and Danio rerio (zebrafish) we identified at least seven tetratricopeptide repeat motifs (TPRs) covering a major part of this protein. TPRs are degenerate 34-amino acid repeat motifs which function as scaffolds mediating protein-protein interactions, often found in multiprotein complexes. In four out of five TPR motifs tested (TPR1, -2, -5, and -6), targeted missense mutagenesis disrupting the motifs at the critical position 8 of each TPR caused complete or partial loss of FANCG function. Loss of function was evident from failure of the mutant proteins to complement the cellular FA phenotype in FA-G lymphoblasts, which was correlated with loss of binding to FANCA. Although the TPR4 mutant fully complemented the cells, it showed a reduced interaction with FANCA, suggesting that this TPR may also be of functional importance. The recognition of FANCG as a typical TPR protein predicts this protein to play a key role in the assembly and/or stabilization of the nuclear FA protein core complex.

Postoperative astigmatism and rotational stability after artisan toric phakic intraocular lens implantation.

PURPOSE: To evaluate deviations in the axis (intended versus achieved) and postoperative astigmatism after implantation of an Artisan toric phakic intraocular lens (IOL). SETTING: University Eye Hospital, Mainz, Germany. METHODS: This prospective study comprised 29 eyes with high ametropia and astigmatism. All eyes had uneventful implantation of a toric phakic IOL through a superior scleral tunnel incision at 12 o'clock. After a minimum of 6 months, the uncorrected visual acuity (UCVA), best correct visual acuity, refraction, and astigmatism were analyzed in all eyes. A multivariate analysis of postoperative astigmatism was performed. RESULTS: After a follow-up of at least 6 months, 95% of eyes were within +/-1.00 diopter (D) of emmetropia and 85% of eyes has a UCVA of 20/30 or better. The difference between the mean intended cylinder axis and achieved cylinder axis was 3.9 degrees (median 3 degrees; range to 13 degrees). The difference between the mean intended axis and the achieved axis between miosis and mydriasis was 1.8 degrees (median 1.5 degrees; range 0 to 5 degrees). The mean postoperative astigmatism after 6 months was 0.56 D with an axis of 31 degrees. Doubled-angle scatterplot analysis showed a tendency toward more flattening in the vertical meridian. CONCLUSIONS: During the 6-month follow-up, no significant rotation was observed after implantation of Artisan toric phakic IOLs to correct high ametropia. A sutureless sclerocorneal superior approach for phakic IOL insertion resulted in moderate to low astigmatism. Induced astigmatism should be taken into consideration during preoperative planning.

Botulinum neurotoxin for pharyngeal constrictor muscle spasm in tracheoesophageal voice restoration.

OBJECTIVES/HYPOTHESIS: The purpose of the study was to evaluate the effectiveness of Botulinum neurotoxin (Botox) for elimination of pharyngeal constrictor muscle spasm in tracheoesophageal voice restoration. STUDY DESIGN: A retrospective review was made of 62 patients between 1991 and 2002 who had Botox as the initial treatment for pharyngeal constrictor muscle spasm. METHODS: One hundred units of Botox properly diluted in 3 mL saline was instilled unilaterally under electromyographic guidance after fluoroscopic identification and marking of the contracted pharyngeal constrictor muscles. The patients were divided into three groups based on their response to the first Botox injection: group I, complete relaxation of the pharyngeal constrictors resulting in fluent voice, intratracheal phonation pressure of 20 to 40 cm H2O, and the ability to say 15 to 20 uninterrupted syllables; group II, hypertonic or incomplete relaxation of the pharyngeal constrictors resulting in intratracheal phonation pressure of 45 to 70 cm H2O and the ability to say 7 or 8 syllables; and group III, failure to produce relaxation of the pharyngeal constrictors. RESULTS: After the first injection of Botox, 49 (79%) patients were in group I or II (41 in group I and 8 in group II) and group III consisted of 13 patients. Thirty-four patients (55%) had group I (28) or II (6) responses for greater than 6 months. A second Botox injection enabled 6 of the 13 failures to move into group I. In all, 8 pharyngeal constrictor muscle myotomies (13%) were ultimately required in the 62 patients. The group I speaker for the longest period has enjoyed 11 years of fluency and successful daily use of a tracheostoma valve after two Botox injections. CONCLUSION: Botox relaxation of the pharyngeal constrictor muscles has proven to be effective, has replaced secondary pharyngeal myotomy for the initial treatment of pharyngeal muscle spasm, and is the only treatment in patients who are not candidates for elective surgery. Radiographic assessment, electromyographically monitored injection, and the number of Botox units appear to be important to successful outcomes.

Assessment of the clinical significance of serum matrix metalloproteinases MMP-2 and MMP-9 in patients with various chronic liver diseases and hepatocellular carcinoma.

Matrix metalloproteinases (MMPs) have the ability to degrade basement membranes and may thus play an important role in extracellular matrix turnover in liver fibrosis and carcinogenesis. Serum levels of MMPs have been suggested as diagnostic markers in these processes. We measured serum MMP-2 and MMP-9 by ELISA in 91 patients with chronic liver disease, including 25 patients with hepatocellular carcinoma (HCC), and in 60 controls. MMP-2 was significantly higher in patients with chronic liver disease compared to controls, and increased with Child-Pugh class. There was a significant correlation between MMP-2 and liver function (bilirubin, albumin, and prothrombin time), and a strong opposite correlation between MMP-9 and these parameters. MMP-2 levels in patients with HCC were significantly higher than in controls, but comparable to patients with chronic liver disease without this malignancy. MMP-9 yielded no significant differences between patients with or without HCC and controls. Serum MMP-2 and to a lesser extent MMP-9 correlate with the severity of liver disease and may reflect changes in extracellular matrix remodeling. Due to a considerable overlap in patients with chronic liver disease with or without HCC, MMP-2 and MMP-9 can not be used as a diagnostic marker for HCC.

Evaluation of the practicality of optical biometry and applanation ultrasound in 253 eyes.

PURPOSE: To evaluate the percentage of eyes that could not be measured using optical biometry and ultrasound applanation and the reasons. SETTING: Department of Ophthalmology, Johannes Gutenberg-University Hospital, Mainz, Germany. METHODS: Optical biometry (IOLMaster, Carl Zeiss Meditec AG) and A-scan ultrasound biometry were performed consecutively in 253 eyes scheduled for cataract surgery the next day. Lens opacities were evaluated with the Opacity Lensmeter (Interzeag), and a slitlamp examination and measurement of visual acuity were performed. The 2 techniques were compared in terms of the rate of and reasons for primary measurement failure. RESULTS: Measurement with the IOLMaster was not possible in 44 eyes (17%). Failed measurements were the result of a combination of low visual acuity and lens opacity in 45% of eyes, posterior subcapsular opacity in 25%, and macular disease in 7%. Measurement with ultrasound biometry was not possible in 10 eyes (4%); 7 eyes were filled with silicone oil and in 3 cases, the patient refused biometry. CONCLUSIONS: Optical biometry allowed comfortable, noncontact, high-precision measurement in the optical axis. Uncorrected visual acuity and lens opacity were predictors of successful measurements. Eyes with dense cataract or poor visual acuity are better evaluated using ultrasound applanation.

Evolutionary clues to the molecular function of fanconi anemia genes.

Fanconi anemia (FA) is an autosomal recessively inherited disease with diverse clinical symptoms including developmental anomalies, predisposition to neoplasia, and a deficiency of hematopoietic stem cells resulting in progressive aplastic anemia. FA is genetically heterogeneous with at least 8 genes being implicated on the basis of functional complementation studies. To date, six FA genes are known: FANCA, FANCC, FANCD2, FANCE, FANCF and FANCG, all of which encode orphan proteins sharing no homology to each other nor to any other known protein. In addition, they do not appear to possess any domains with homology to currently known protein domains, which makes a prediction about their molecular action difficult. Studying the molecular evolution of FA genes and their products using sensitive database search methods such as PSI-BLAST may provide novel insight into the nature of the FA pathway and its relationship to hematopoiesis, embryonic development and the origin of malignancies. Preliminary results of such an approach show that at least one FA protein, FANCG, may contain a known domain, suggesting that this protein is a member of the family of tetratricopeptide repeat-containing proteins.

Characterization, expression and complex formation of the murine Fanconi anaemia gene product Fancg.

BACKGROUND: Fanconi anaemia (FA) is an autosomal recessive chromosomal instability disorder. Six distinct FA disease genes have been identified, the products of which function in an integrated pathway that is thought to support a nuclear caretaker function. Comparison of FA gene characteristics in different species may help to unravel the molecular function of the FA pathway. RESULTS: We have cloned the murine homologue of the Fanconi anaemia complementation group G gene, FANCG/XRCC9. The murine Fancg protein shows an 83% similarity to the human protein sequence, and has a predicted molecular weight of 68.5 kDa. Expression of mouse Fancg in human FA-G lymphoblasts fully corrects their cross-linker hypersensitivity. At mRNA and protein levels we detected the co-expression of Fancg and Fanca in murine tissues. In addition, mouse Fancg and Fanca proteins co-purify by immunoprecipitation. Upon transfection into Fanca-deficient mouse embryonic fibroblasts EGFP-Fancg chimeric protein was detectable in the nucleus. CONCLUSIONS: We identified a murine cDNA, Fancg, which cross-complements the cellular defect of human FA-G cells and thus represents a true homologue of human FANCG. Spleen, thymus and testis showed the highest Fancg expression levels. Although Fancg and Fanca are able to form a complex, this interaction is not required for Fancg to accumulate in the nuclear compartment.