A Novel Methylation PCR that Offers Standardized Determination of FMR1 Methylation and CGG Repeat Length without Southern Blot Analysis

Fragile X syndrome and associated disorders are characterized by the number of CGG repeats and methylation status of the FMR1 gene for which Southern blot (SB) historically has been required for analysis. This study describes a simple PCR-only workflow (mPCR) to replace SB analysis, that incorporates novel procedural controls, treatment of the DNA in separate control and methylation-sensitive restriction endonuclease reactions, amplification with labeled primers, and two-color amplicon sizing by capillary electrophoresis. mPCR was evaluated in two independent laboratories with 76 residual clinical samples that represented typical and challenging fragile X alleles in both males and females. mPCR enabled superior size resolution and analytical sensitivity for size and methylation mosaicism compared to SB. Full mutation mosaicism was detected down to 1% in a background of 99% normal allele with 50- to 100-fold less DNA than required for SB. A low level of full mutation mosaicism in one sample was detected using mPCR but not observed using SB. Overall, the sensitivity for detection of full mutation alleles was 100% (95% CI: 89%–100%) with an accuracy of 99% (95% CI: 93%–100%). mPCR analysis of DNA from individuals with Klinefelter and Turner syndromes, and DNA from sperm and blood, were consistent with SB. As such, mPCR enables accurate, sensitive, and standardized methods of FMR1 analysis that can harmonize results across different laboratories.Diverse developmental, mental, and reproductive disorders are associated with both the number of cytosine-guanine-guanine (CGG) repeats and the methylation status of the fragile X mental retardation-1 (FMR1, {"type":"entrez-nucleotide","attrs":{"text":"NM_002024.4","term_id":"169881241","term_text":"NM_002024.4"}}NM_002024.4) gene.^1–3 Excessive CGG repeat expansion is directly linked with hypermethylation of the gene through an epigenetic mechanism that is distinct from X chromosome inactivation and developmentally timed after lyonization.⁴ Because the FMR1 protein (FMRP) is a master regulator of genes involved in synaptic plasticity,⁵ the intellectual and behavioral consequences of quantitative FMR1 silencing are profound. Methylation of full mutation expansions (>200 CGG), however, can be incomplete, and less severe phenotypes may be associated with methylation mosaicism.^6–8 In premutation alleles (55 to 200 CGG) the number of CGG can influence the risks and phenotype of fragile X–associated tremor/ataxia syndrome (FXTAS, OMIM 300623),⁹ fragile X–associated primary ovarian insufficiency (FXPOI, OMIM 300624),^10,11 and autism spectrum disorders.^12,13 Methylation status or X-inactivation in females may further influence the risk and phenotype of these conditions even if the results reported are still inconclusive.^10,11,14 These premutation alleles are relatively common in the general population, occurring in 1 in 130 to 250 women and in 1 in 250 to 810 men, as reported in the United States,^15,16 suggesting a broader need for FMR1 characterization in the general population. Differences in methylation status have also been reported between DNA from whole blood compared to skin fibroblasts, which may be closer in cellular origin to brain and more reflective of phenotype.¹⁷ Thus, it is critical to accurately and reliably assess the CGG repeat length and spectrum of methylation characteristics in individuals with FMR1 premutation and full mutation expansions, and to enable analysis of alternative sample types rather than peripheral blood.Southern blot (SB) analysis is currently the gold standard method for determining size and methylation status in expanded FMR1 alleles. However, this procedure is severely limited by the amount of genomic DNA material that is required, a tedious workflow, and variable sensitivity. Disadvantages of SB include low resolution and the inability to accurately size premutation and normal alleles. Therefore, most clinical laboratories currently rely on a combination of PCR and SB analysis because of the technical limitations and the specific pitfalls of each method that, if used alone, could induce potential misinterpretation of the genotype.^18,19Various alternatives to SB analysis have been reported that use bisulfite or enzymatic pretreatment of DNA before PCR to obtain methylation status.^20–25 These methods have been typically restricted to the analysis of male samples because of the inefficiency of PCR or confounding presence of two X chromosomes in females.^24,25 Alternative methylation markers have been proposed but lack direct association with the number of CGG repeats.²⁶ Concurrent assessment of CGG repeat length and of allele-specific methylation status in both males and females has been demonstrated.²³ This methylation PCR (mPCR) method was based on the analysis of DNA treated with methylation-sensitive endonucleases before FMR1 gene-specific PCR.²⁷ The results were concordant with SB analysis across a range of genotypes. However, a reference control was incorporated that overlapped with samples having alleles of 38 to 42 CGG, and the approach lacked additional controls that would benefit routine testing in a clinical laboratory environment.Herein, we report the inclusion of novel procedural controls and a simplified workflow for mPCR that advance FMR1 analysis without the need for SB analysis. We compare results between methods using a range of challenging clinical samples obtained from two European laboratories. We demonstrate concordance and improved detection of methylation and size mosaicism relative to SB analysis. The ability to analyze novel sample types and samples with aneuploidy that might be encountered during standard fragile X testing is presented. Consequently, this report provides the first interlaboratory validation of a PCR-based FMR1 assay that can accurately assess repeat length and methylation status in both males and females (including all premutation and full mutation alleles), and thus support routine fragile X testing without the requirement for SB analysis.     

Biphasic alveolosquamoid renal carcinoma: a histomorphological,immunohistochemical, molecular genetic,and ultrastructural study of a distinctive morphologic variant of renal cell carcinoma

Only a few cases of sarcomatoid renal cell carcinomas (RCCs) with squamous differentiation have been published. We present 2 RCCs exhibiting a hitherto not reported biphasic neoplastic cell population exhibiting a predominantly alveolar architecture where squamoid differentiation was identified in one of the neoplastic cell populations. None of the tumors showed chromophobe features or any evidence of sarcomatoid transformation. The tumors arose in 2 adult patients and were characterized by routine histology, immunohistochemistry, ultrastructure, array comparative genomic hybridization, confirmatory fluorescent in situ hybridization, and loss of heterozygosity analysis. Tumors measured 3 and 4 cm and were located within the renal parenchyma and had no pelvicalyceal connection. Both tumors were composed of a distinctly dual-cell population. The larger tumor cells displayed squamoid features and formed round well-demarcated solid alveolated islands that, in large parts, were surrounded by a smaller neoplastic cell component. The squamoid cells were immunoreactive for cytokeratins (CKs) (AE1-AE3, Cam 5.2, CK5/6, CK7, and CK20), epithelial membrane antigen, racemase/AMACR, and carboanhydrase IX (in 1 case focally). The small cell population was positive for CK7, epithelial membrane antigen, and racemase/AMACR, whereas CK20, AE1-3, and carboanhydrase IX were negative. CD10 was focally positive in the large squamoid cells in 1 case. Cathepsin K, E-cadherin, and CD117 displayed focal positivity in 1 case. Vimentin, RCC marker, parvalbumin, S100 protein, S100 A1, p63, p53, CDX2, uroplakin III, HMB45, TFE3, WT1, synaptophysin, chromogranin A, thyroglobulin, and TTF1 were negative. The proliferative activity (Ki-67) was low (1%) in the small cell component in both cases, whereas the large neoplastic tumor cells displayed a significantly higher proliferation (20%-35%). Ultrastructurally, desmosomes and tonofilaments were identified in the large tumor cells, confirming squamoid differentiation in a subset of tumor cells. Array comparative genomic hybridization of 1 analyzable case (confirmed with fluorescent in situ hybridization and loss of heterozygosity analysis) revealed partial or complete losses of chromosomes 2, 5, 6, 9, 12, 15, 16, 17, 18 and 22, (including biallelic loss of CDKN2A locus) and partial gains of chromosomes 1, 5, 11, 12 and 13. Follow-up at 6 years showed no recurrence or metastasis in 1 patient. The other (male) patients had a subcutaneous metastasis at presentation, but during a 1-year follow-up no evidence of recurrence or further metastatic events have been documented. Our data indicate that biphasic alveolosquamoid renal carcinoma is a unique and distinctive tumor. The large squamoid and small tumor cells have overlapping but still distinctive immunohistochemical patterns of protein expression. Multiple chromosomal aberrations were identified, some of them located in regions with known tumor suppressor genes and oncogenes.     

Isokinetic dynamometry applied to shoulder rotators – Velocity limitations in eccentric evaluations

The objectives of this study were to evaluate if collegiate overhead athletes, with and without shoulder pain, and non-athletes could reach a preset velocity in internal and external shoulder rotation isokinetic evaluations; and to evaluate the correlation between torque and velocity. Controlled laboratory study, cross-sectional. Evaluations were performed using the isokinetic dynamometer Biodex System 3. Participants were assessed seated, with the arm at 90° of shoulder abduction and 90° of elbow flexion, from neutral rotation to 90° of external rotation. Five maximal contractions of isokinetic concentric and reactive eccentric internal and external rotation were performed at the velocities 90°/s, 180°/s and 240°/s. Data were processed with using MatLab. Most participants did not reach the isokinetic phase during eccentric tests at 180°/s and 240°/s, particularly in the external rotators evaluation. High correlations between torque and velocity of eccentric tests were found. The groups presented no differences in maximal velocity attained in trials which preset velocity was not reached. These results call into question the use of reactive eccentric tests at velocities higher than 180°/s for the isokinetic evaluation of shoulder external rotators in collegiate overhead athletes and non-athletes in this specific position. In such cases, careful evaluation of the velocity is recommended to determine if the isokinetic phase was reached.     

Lymphocele and renal transplantation

Lymphoceles are a well-known surgical complication of kidney transplantation. We retrospectively analyzed patients with lymphoceles among our renal transplant recipients. During the last 39 years, we performed 922 renal transplantations. Lymphoceles were diagnosed and treated in 45 (4.9%) patients. We used the following methods: percutaneous drainage with instillation of povidone-iodide in 36 (80%), percutaneous drainage with instillation of tetracycline in one (2.2%), percutaneous aspiration in four (8.9%) and surgical treatment in four (8.9%) patients. In all four (8.9%) patients with relapse, secondary procedure was successful. In total, open surgery was done in five (11.1%) and laparoscopy in four (8.9%) patients. Percutaneous drainage of lymphoceles, with or without the instillation of a sclerosant, is the first-line treatment. Laparoscopic fenestration of lymphoceles has become an alternative to percutaneous drainage, especially in case of post-drainage relapse.     

Simultaneous Chlamydia trachomatis and HPV infection in pregnant women

Pregnancy is associated with HPV infection and with Chlamydia trachomatis (CT) infection mostly due to the natural immunosuppression. In addition, pregnancy associated to CT infection can lead to adverse conditions to the woman and fetus, and CT is also believed to be a co‐factor in human immunodeficiency virus infection and HPV‐induced cervical cancer. The aim of this study was to establish the odds ratios (OR) of CT infection in to HPV‐infected pregnant women and vice versa of women stratified by age (<25 years) and marital status. This work is part of a national multicentric transversal study carried out in six Brazilian cities supported by the Ministry of Health of Federal Government of Brazil in 2003. Cervical scrapes of 371 pregnant women were sampled. We performed a hybrid capture‐2 technique to diagnose these samples on HPV and CT infection, and the women responded a questionnaire. Significant association was observed between nonstable marital status and hr‐HPV infection [OR = 2.61 (1.38–4.97) P = 0.003)], and age <25 years old [OR = 2.26 (1.09–4.71) P = 0.029]. Nonstable marital status was also associated with lr‐HPV infection [OR = 2.67 (1.59–4.50) P < 0.001), and age <25 years old [OR = 2.55 (1.51–4.32) P < 0.001). Fifty of the 371 pregnant women were infected with hr‐HPV (13.5%) and 111 (30.0%) were infected with lr‐HPV. The coinfections of HPV and CT were found in 31 women, that is, 8.36% of the pregnant women (P < 0.001). The high rate of simultaneous CT and HPV infection in pregnant women favors the recommendation to screen pregnant women for both CT and HPV. Diagn. Cytopathol. 2010. © 2009 Wiley‐Liss, Inc.     

Microperimetry in diabetic retinopathy

Diabetic retinopathy has an enormous impact on visual function, even before permanent visual acuity loss. Moreover, adequate functional tests are mandatory to diagnose and follow diabetic patients treated for diabetic macular edema (DME). More precisely, the visual function safety profile of any therapy for DME should be accurately investigated. Microperimetry offers the possibility to obtain an exact fundus-related quantification of retinal sensitivity, and it is changing the current approach to the functional investigation of diabetic retinopathy.