Could the Leukocyte X Chromosome Inactivation Pattern Be Extrapolated to Hair Bulbs?

Background: The androgen receptor gene is located on the X chromosome with a polymorphic tract of CAG repeats that is inversely correlated to the receptor`s transactivation activity. A short CAG tract is associated with hyperandrogenic disorders. In women, one of the X chromosomes is inactivated and the X chromosome inactivation (XCI) pattern varies among tissues. Previous studies of hyperandrogenic disorders only evaluated XCI in leukocytes. Objective: To evaluate whether the XCI pattern in leukocytes could be extrapolated to those in hair bulbs. Material: A total of 58 healthy women were used for this study. DNA was extracted from leukocytes (n = 58 women) and pubic (n = 53 women) and scalp hair (n = 21 women). Methods: Hpa II digested and undigested DNA samples underwent fluorescence PCR GeneScan (R) analysis. Results: A significant and positive correlation of XCI was found between leukocytes and hair bulbs. However, individual comparisons showed that 13 and 19% of the women presented a different leukocyte XCI pattern in pubic hair and similar in leukocytes and hair bulbs of normal women indicating that leukocyte DNA is useful for XCI analysis. However, the XCI pattern could vary among tissues from the same subject, indicating that care should be taken when extrapolating individual leukocyte XCI patterns to other tissue. Copyright (C) 2010 S. Karger AG, Basel

The common p450 oxidoreductase variant A503V is not a modifier gene for 21-hydroxylase deficiency

Context: 21-hydroxylase deficiency (21OHD) is a common genetic disorder caused by mutations in the CYP21A2 gene, which encodes the adrenal 21-hydroxylase, microsomal P450c21. CYP21A2 gene mutations generally correlate well with impaired P450c21 enzymatic activity and the clinical findings in 21OHD, but occasional discrepancies between genotype and phenotype suggest the effects of modifier genes. Mutations in P450 oxidoreductase (POR), the protein that transfers electrons from reduced nicotinamide adenine dinucleotide phosphate to all microsomal P450s, can ameliorate the 21OHD phenotype and, therefore, could be a modifier gene. Objectives: We sought to identify POR variants in patients with 21OHD having discordant phenotype and genotype, and to evaluate their effect on 21-hydroxylase activity. Patients and Methods: We determined the CYP21A2 genotypes of 313 Brazilian patients with 21OHD and correlated the genotype and phenotype. The POR gene was sequenced in 17 patients with discordant genotype and phenotype. Wild-type and A503V POR, and P450c21 were expressed in bacteria and reconstituted in vitro. Activities were assayed by conversion of [C-14] progesterone to deoxycorticosterone and [H-3]17-hydroxyprogesterone to 11-deoxycortisol, and assessed by thin layer chromatography and phosphorimaging. Results: The A503V POR variant was found in 10 of 30 alleles, the same ratio as in the normal population. There were no significant differences in Michaelis constant, maximum velocity and maximum velocity/Michaelis constant of 21-hydroxylase activity supported by wild-type and A503V POR. Conclusion: The only POR missense polymorphism found in atypical 21OHD patients was A503V. Although A503V reduces P450c17 enzymatic activity, it does not influence P450c21 activity, indicating that POR A503V does not modify the 21OHD phenotype.

Heterozygosity for the S180L Variant of MAL/TIRAP, a Gene Expressing an Adaptor Protein in the Toll-Like Receptor Pathway, Is Associated with Lower Risk of Developing Chronic Chagas Cardiomyopathy

Background. Chagas disease is caused by the protozoan parasite Trypanosoma cruzi. Among T. cruzi-infected individuals, only a subgroup develops severe chronic Chagas cardiomyopathy (CCC); the majority remain asymptomatic. T. cruzi displays numerous ligands for the Toll-like receptors (TLRs), which are an important component of innate immunity that lead to the transcription of proinflammatory cytokines by nuclear factor-kappa B. Because proinflammatory cytokines play an important role in CCC, we hypothesized that single-nucleotide polymorphisms (SNPs) in the genes that encode proteins in the TLR pathway could explain differential susceptibility to CCC among T. cruzi-infected individuals. Methods. For 169 patients with CCC and 76 T. cruzi-infected, asymptomatic individuals, we analyzed SNPs by use of polymerase chain reaction-restriction fragment length polymorphism analysis for the genes TLR1, TLR2, TLR4, TLR5, TLR9, and MAL/TIRAP, which encodes an adaptor protein. Results. Heterozygous carriers of the MAL/TIRAP variant S180L were more prevalent in the asymptomatic group (24 [32%] of 76 subjects) than in the CCC group (21 [12%] of 169) (chi(2) = 12.6; P = .0004 [adjusted P (P(c)) = .0084]; odds ratio [OR], 0.31 [95% confidence interval {CI}, 0.16-0.60]). Subgroup analysis showed a stronger association when asymptomatic patients were compared with patients who had severe CCC (i.e., patients with left-ventricular ejection fraction <= 40%) (chi(2) = 11.3; P = .0008 [P(c) = .017]; OR, 0.22 [95% CI, 0.09-0.56]) than when asymptomatic patients were compared with patients who had mild CCC (i.e., patients with left-ventricular ejection fraction >40%) (chi(2) = 7.7; P = .005 [P(c) = .11]; OR, 0.33 [95% CI, 0.15-0.73]). Conclusion. T. cruzi-infected individuals who are heterozygous for the MAL/TIRAP S180L variant that leads to a decrease in signal transduction upon ligation of TLR2 or TLR4 to their respective ligand may have a lower risk of developing CCC.

Construction of a 1-Mb restriction-mapped cosmid contig containing the candidate region for the familial mediterranean fever locus (MEFV) on chromosome 16p13.3

In this paper we describe the assembly and restriction map of a 1.05-Mb cosmid contig spanning the candidate region for familial Mediterranean fever (FMF), a recessively inherited disorder of inflammation localized to 16p13.3. Using a combination of cosmid walking and screening for P1, PAC, BAG, and YAC clones, we have generated a contig of genomic clones spanning similar to 1050 kb that contains the FMF critical region. The map consists of 179 cosmid, 15 P1, 10 PAC, 3 BAG, and 17 YAC clones, anchored by 27 STS markers. Eight additional STSs have been developed from the similar to 700 kb immediately centromeric to this genomic region. Five of the 35 STSs are microsatellites that have not been previously reported. NotI and EcoRI mapping of the overlapping cosmids, hybridization of restriction fragments from cosmids to one another, and STS analyses have been used to validate the assembly of the contig. Our contig totally subsumes the 250-kb interval recently reported, by founder haplotype analysis, to contain the FMF gene. Thus, our high-resolution clone map provides an ideal resource for transcriptional mapping toward the eventual identification of this disease gene. (C) 1997 Academic Press.

Genetic heterogeneity in familial acute myelogenous leukemia: Evidence for a second locus at chromosome 16q21-23.2

The identification of genes responsible for the rare cases of familial leukemia may afford insight into the mechanism underlying the more common sporadic occurrences. Here we test a single family with 11 relevant meioses transmitting autosomal dominant acute myelogenous leukemia (AML) and myelodysplasia for linkage to three potential candidate loci. In a different family with inherited AML, linkage to chromosome 21q22.1-22.2 was recently reported; we exclude linkage to 21q22.1-22.2, demonstrating that familial AML is a heterogeneous disease. After reviewing familial leukemia and observing anticipation in the form of a declining age of onset with each generation, we had proposed 9p21-22 and 16q22 as additional candidate loci. Whereas linkage to 9p21-22 can be excluded, the finding of a maximum two-point LOD score of 2.82 with the microsatellite marker D16S522 at a recombination fraction theta = 0 provides evidence supporting linkage to 16q22. Haplotype analysis reveals a 23.5-cM (17.9-Mb) commonly inherited region among all affected family members extending from D16S451 to D1GS289, In order to extract maximum linkage information with missing individuals, incomplete informativeness with individual markers in this interval, and possible deviance from strict autosomal dominant inheritance, we performed nonparametric linkage analysis (NPL) and found a maximum NPL statistic corresponding to a P-value of .00098, close to the maximum conditional probability of linkage expected for a pedigree with this structure. Mutational analysis in this region specifically excludes expansion of the AT-rich minisatellite repeat FRA16B fragile site and the CAG trinucleotide repeat in the E2F-4 transcription factor. The ''repeat expansion detection'' method, capable of detecting dynamic mutation associated with anticipation, more generally excludes large CAG repeat expansion as a cause of leukemia in this family.

Differences Between ""Classical"" Risk Factors for Infections Caused by Methicillin-Resistant Staphylococcus aureus (MRSA) and Risk Factors for Nosocomial Bloodstream Infections Caused by Multiple Clones of the Staphylococcal Cassette Chromosome mec Type IV MRSA Strain

OBJECTIVE. To identify risk factors associated with nosocomial bloodstream infections caused by multiple clones of the staphylococcal cassette chromosome mec (SCCmec) type IV strain of methicillin-resistant Staphylococcus aureus (MRSA). DESIGN. An unmatched case-control study (at a ratio of 1 : 2) performed during the period from October 2002 through September 2003. SETTING. A 2,000-bed tertiary care teaching hospital affiliated with the University of Sao Paulo in Sao Paulo, Brazil. METHODS. Case patients (n = 30) were defined either as patients who had a bloodstream infection due to SCCmec type IV strains of MRSA diagnosed at least 48 hours after hospital admission or as neonates with the infection who were born in the hospital. Control patients (n = 60) were defined as patients with SCCmec type III MRSA infection diagnosed at least 48 hours after hospital admission. Genes n = 60 encoding virulence factors were studied in the isolates recovered from case patients, and molecular typing of the SCCmec type IV MRSA isolates was also done by pulsed-field gel electrophoresis and multilocus sequence typing. RESULTS. In multivariate analysis, the following 3 variables were significantly associated with having a nosocomial bloodstream infection caused by SCCmec type IV strains of MRSA: an age of less than 1 year, less frequent use of a central venous catheter (odds ratio [OR], 0.07 [95% confidence interval {CI}, 0.02-0.28]; P = .001), and female sex. A second analysis was performed that excluded the case and Pp. 001 control patients from the neonatal unit, and, in multivariate analysis, the following variables were significantly associated with having a nosocomial bloodstream infection caused by SCCmec type IV strains of MRSA: less frequent use of a central venous catheter (OR, 0.12 [95% CI, 0.03-0.55]; P = .007), lower Acute Physiology and Chronic Health Evaluation II score on admission (OR, 0.14 [95% CI, 0.03-0.61];), less frequent surgery (OR, 0.21 [95% CI, 0.06-0.83];), and female sex (OR, 5.70 [95% CI, 1.32-24.66]; P =.020). P = .009 Pp. 025 Pp). Of the 29 SCCmec type IV MRSA isolates recovered from case patients, none contained the Panton-Valentine leukocidin, gamma-hemolysin, enterotoxin B or C, or toxic shock syndrome toxin-1. All of the isolates contained genes for the LukE-LukD leukocidin and alpha-hemolysin. Genes for enterotoxin A were present in 1 isolate, and genes for beta-hemolysin were present in 3 isolates. CONCLUSIONS. ""Classical"" risk factors do not apply to patients infected with the SCCmec type IV strain of MRSA, which is an important cause of nosocomial bacteremia. This strain infects a patient population that is less ill and has had less frequent invasive procedures than a patient population infected with the multidrug-resistant strain of SCCmec type III MRSA. We found that virulence factors were rare and that Panton-Valentine leukocidin was absent. There were multiple clones of the SCCmec type IV strain in our hospital. Children under 1 year of age were at a higher risk. There was a predominant clone ( sequence type 5) in this patient population.

Recessive COL6A2 C-globular Missense Mutations in Ullrich Congenital Muscular Dystrophy ROLE OF THE C2a SPLICE VARIANT

Ullrich congenital muscular dystrophy (UCMD) is a disabling and life-threatening disorder resulting from either recessive or dominant mutations in genes encoding collagen VI. Although the majority of the recessive UCMD cases have frameshift or nonsense mutations in COL6A1, COL6A2, or COL6A3, recessive structural mutations in the COL6A2 C-globular region are emerging also. However, the underlying molecular mechanisms have remained elusive. Here we identified a homozygous COL6A2 E624K mutation (C1 subdomain) and a homozygous COL6A2 R876S mutation (C2 subdomain) in two UCMD patients. The consequences of the mutations were investigated using fibroblasts from patients and cells stably transfected with the mutant constructs. In contrast to expectations based on the clinical severity of these two patients, secretion and assembly of collagen VI were moderately affected by the E624K mutation but severely impaired by the R876S substitution. The E624K substitution altered the electrostatic potential of the region surrounding the metal ion-dependent adhesion site, resulting in a collagen VI network containing thick fibrils and spots with densely packed microfibrils. The R876S mutation prevented the chain from assembling into triple-helical collagen VI molecules. The minute amount of collagen VI secreted by the R876S fibroblasts was solely composed of a faster migrating chain corresponding to the C2a splice variant with an alternative C2 subdomain. In transfected cells, the C2a splice variant was able to assemble into short microfibrils. Together, the results suggest that the C2a splice variant may functionally compensate for the loss of the normal COL6A2 chain when mutations occur in the C2 subdomain.

Association between a 15q25 gene variant, smoking quantity and tobacco-related cancers among 17 000 individuals

Methods We performed a detailed analysis of one 15q single nucleotide polymorphism (SNP) (rs16969968) with smoking behaviour and cancer risk in a total of 17 300 subjects from five LC studies and four upper aerodigestive tract (UADT) cancer studies. Results Subjects with one minor allele smoked on average 0.3 cigarettes per day (CPD) more, whereas subjects with the homozygous minor AA genotype smoked on average 1.2 CPD more than subjects with a GG genotype (P < 0.001). The variant was associated with heavy smoking (> 20 CPD) [odds ratio (OR) = 1.13, 95% confidence interval (CI) 0.96-1.34, P = 0.13 for heterozygotes and 1.81, 95% CI 1.39-2.35 for homozygotes, P < 0.0001]. The strong association between the variant and LC risk (OR = 1.30, 95% CI 1.23-1.38, P = 1 x 10(-18)), was virtually unchanged after adjusting for this smoking association (smoking adjusted OR = 1.27, 95% CI 1.19-1.35, P = 5 x 10(-13)). Furthermore, we found an association between the variant allele and an earlier age of LC onset (P = 0.02). The association was also noted in UADT cancers (OR = 1.08, 95% CI 1.01-1.15, P = 0.02). Genome wide association (GWA) analysis of over 300 000 SNPs on 11 219 subjects did not identify any additional variants related to smoking behaviour. Conclusions This study confirms the strong association between 15q gene variants and LC and shows an independent association with smoking quantity, as well as an association with UADT cancers.

A Sex-Specific Association between a 15q25 Variant and Upper Aerodigestive Tract Cancers

Background: Sequence variants located at 15q25 have been associated with lung cancer and propensity to smoke. We recently reported an association between rs16969968 and risk of upper aerodigestive tract (UADT) cancers (oral cavity, oropharynx, hypopharynx, larynx, and esophagus) in women (OR = 1.24, P = 0.003) with little effect in men (OR = 1.04, P = 0.35). Methods: In a coordinated genotyping study within the International Head and Neck Cancer Epidemiology (INHANCE) consortium, we have sought to replicate these findings in an additional 4,604 cases and 6,239 controls from 10 independent UADT cancer case-control studies. Results: rs16969968 was again associated with UADT cancers in women (OR = 1.21, 95% CI = 1.08-1.36, P = 0.001) and a similar lack of observed effect in men [OR = 1.02, 95% CI = 0.95-1.09, P = 0.66; P-heterogeneity (P(het)) = 0.01]. In a pooled analysis of the original and current studies, totaling 8,572 UADT cancer cases and 11,558 controls, the association was observed among females (OR = 1.22, 95% CI = 1.12-1.34, P = 7 x 10(-6)) but not males (OR = 1.02, 95% CI = 0.97-1.08, P = 0.35; P(het) = 6 x 10(-4)). There was little evidence for a sex difference in the association between this variant and cigarettes smoked per day, with male and female rs16969968 variant carriers smoking approximately the same amount more in the 11,991 ever smokers in the pooled analysis of the 14 studies (P(het) = 0.86). Conclusions: This study has confirmed a sex difference in the association between the 15q25 variant rs16969968 and UADT cancers. Impact: Further research is warranted to elucidate the mechanisms underlying these observations. Cancer Epidemiol Biomarkers Prev; 20(4); 658-64. (C) 2011 AACR.

Development of Y-chromosome-Specific SCAR Markers Conserved in Taurine, Zebu and Bubaline Cattle

Contents Sex pre-selection of bovine offsprings has commercial relevance for cattle breeders and several methods have been used for embryo sex determination. Polymerase chain reaction (PCR) has proven to be a reliable procedure for accomplishing embryo sexing. To date, most of the PCR-specific primers are derived from the few single-copy Y-chromosome-specific gene sequences already identified in bovines. Their detection demands higher amounts of embryonic genomic material or a nested amplification reaction. In order to circumvent this, limitation we searched for new male-specific sequences potentially useful in embryo sexing using random amplified polymorphic DNA (RAPD) analysis. Random amplified polymorphic DNA (RAPD) assay reproducibility problems can be overcome by its conversion into Sequence Characterized Amplified Region (SCAR) markers. In this work, we describe the identification of two bovine male-specific markers (OPC16(323) and OPF10(1168)) by means of RAPD. These markers were successfully converted into SCARs (OPC16(726) and OPF10(984)) using two pairs of specific primers.Furthermore, inverse PCR (iPCR) methodology was successfully applied to elongate OPC16(323) marker in 159% (from 323 to 837 bp). Both markers are shown to be highly conserved (similarity >= 95%) among bovine zebu and taurine cattle; OPC16(323) is also highly similar to a bubaline Y-chromosome-specific sequence. The primers derived from the two Y-chromosome-specific conserved sequences described in this article showed 100% accuracy when used for identifying male and female bovine genomic DNA, thereby proving their potential usefulness for bovine embryo sexing.

A new morphological variant of uterine PEComas with sex-cord-like pattern and WT1 expression: more doubts about the existence of uterine PEComas

PEComas are rare neoplasms that are sometimes associated with the tuberous sclerosis complex. They typically contain perivascular epithelioid cells that coexpress muscle and melanocytic markers. However, apart from these classical features, considerable clinical, pathologic, and immunohistochemical variation has been reported. WT1, the Wilms tumor gene product, can be expressed in various tumors from different anatomical sites, including sex-cord and other ovarian tumors with a sertoliform pattern. Neither a sex-cord like pattern nor WT1 expression has been described in PEComas. Here, we describe a case of uterine PEComa with a pattern of infiltration into the myometrium that is similar to stromal sarcomas, characterized by tongues and endovascular growing. The architecture and cellular morphology were similar to sex-cord tumors, and the PEComa was diffusely and strongly positive for WT1. We reviewed, from our files, an additional 9 cases of PEComa from different sites, and found WT1 expression in one more soft tissue tumor. We discuss the relationship between PEComas and other uterine sarcomas. (C) 2010 Elsevier Inc. All rights reserved.

Cytogenetic and Molecular Evaluation and 20-Year Follow-Up of a Patient With Ring Chromosome 14

We present a 20-year follow-up on a patient with a ring chromosome 14. The ring chromosome was studied by fluorescence in-situ hybridization (FISH), multiplex-ligation probe amplification (MLPA), and genome wide SNP array, and no deletions of chromosome 14 were detected, although the telomeric repeat sequence was absent from the ring chromosome. The patient had skeletal abnormalities, and susceptibility to infections, as well as seizures and retinal pigmentation, which are commonly found in individuals with a ring 14. Our patient corroborates the idea that even when no genes are lost during ring formation, a complete ring chromosome can produce phenotypic alterations, which presumably result from ring instability or gene silencing due to the new chromosomal architecture. (C) 2010 Wiley-Liss, Inc.

Genetic association and cellular function of MC1R variant alleles in human pigmentation

We have examined MC1R variant allele frequencies in the general population of South East Queensland and in a collection of adolescent dizygotic and monozygotic twins and family members to define statistical associations with hair and skin color, freckling, and mole count. Results of these studies are consistent with a linear recessive allelic model with multiplicative penetrance in the inheritance of red hair. Four alleles, D84E, R151C, R160W, and D294H, are strongly associated with red hair and fair skin with multinomial regression analysis showing odds ratios of 63, 118, 50, and 94, respectively. An additional three low-penetrance alleles V60L, V92M, and R163Q have odds ratios 6, 5, and 2 relative to the wild-type allele. To address the cellular effects of MC1R variant alleles in signal transduction, we expressed these receptors in permanently transfected HEK293 cells. Measurement of receptor activity via induction of a cAMP-responsive luciferase reporter gene found that the R151C and R160W receptors were active in the presence of NDP-MSH ligand, but at much reduced levels compared with that seen with the wild-type receptor. The ability to stimulate phosphorylation of the cAMP response element binding protein (CREB) transcription factor was also apparent in all stimulated MC1R variant allele-expressing HEK293 cell extracts as assessed by immunoblotting. In contrast, human melanoma cell lines showed wide variation in the their ability to undergo cAMP-mediated CREB phosphorylation. Culture of human melanocytes of known MC1R genotype may provide the best experimental approach to examine the functional consequences for each MC1R variant allele. With this objective, we have established more than 300 melanocyte cell strains of defined MC1R genotype.

Microduplication of the ICR2 Domain at Chromosome 11p15 and Familial Silver-Russell Syndrome

Silver-Russell syndrome (SRS) is characterized by severe intrauterine and postnatal growth retardation in association with a typical small triangular face and other variable features. Genetic and epigenetic disturbances are detected in about 50% of the patients. Most frequently, SRS is caused by altered gene expression on chromosome 11p15 due to hypomethylation of the telomeric imprinting center (ICR1) that is present in at least 40% of the patients. Maternally inherited duplications encompassing ICR1 and ICR2 domains at 11p15 were found in a few patients, and a microduplication restricted to ICR2 was described in a single SRS child. We report on a microduplication of the ICR2 domain encompassing the KCNQ1, KCNQ1OT1, and CDKN1C genes in a three-generation family: there were four instances of paternal transmissions of the microduplication from a single male uniformly resulting in normal offspring, and five maternal transmissions, via two clinically normal sisters, with all the children exhibiting SRS. This report provides confirmatory evidence that a microduplication restricted to the ICR2 domain results in SRS when maternally transmitted. (C) 2011 Wiley-Liss, Inc.