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PubMed2003

The correlation between aberrant connexin 43 mRNA expression induced by promoter methylation and nodal micrometastasis in non-small cell lung cancer.

Reduced connexin (Cx) 43 gene expression has been shown in most of lung tumors and cancer cell lines. Although aberrant Cx43 gene expression was linked with lung tumorigenesis, our understanding to the mechanism was still limited. We hypothesized that the evidence of aberrant Cx43 gene expression was gradually intensified from adjacent normal lung tissues surrounding tumors toward tumor tissues. In this study, 90 lung tumors and adjacent normal tissues were collected to examine Cx43 mRNA expression by reverse transcription-PCR (RT-PCR). Our data showed that Cx43 mRNA expression in adjacent normal lung tissue was significantly correlated with nodal involvement (P = 0.03), but the similar trend was not observed in tumor tissues. To verify whether lack of Cx43 mRNA expression resulted from promoter methylation, PCR-based methylation assay was performed for Cx43 promoter methylation analysis. A higher frequency of promoter methylation was observed in Cx43 mRNA-negative patients (21 of 33, 63.7%) compared with Cx43 mRNA-positive patients (3 of 57, 5.3%, P < 0.0001). To elucidate whether aberrant Cx43 gene expression originated from adjacent normal lung tissues, 25 lung tumors and each of five adjacent normal tissues at various distances from tumor tissues were collected to examine Cx43 mRNA and protein expression by RT-PCR and Western blot, respectively. The results show that Cx43 mRNA and protein expressions gradually decreased from adjacent normal lung tissues to tumor tissues with a positive correlation to the distance from the tumor tissues. Gel-shift assay data also revealed that shifted band binding with AP1 was only observed in adjacent normal tissues, which were far from the tumor tissues. These results indicate that promoter methylation may interfere with AP1 binding to the promoter to cause aberrant Cx43 gene expression. Thus, Cx43 mRNA in adjacent normal tissue surrounding lung tumor simply detected by RT-PCR may act as a molecular marker of nodal micrometastasis in non-small cell lung cancer.

Adenocarcinoma↗

PubMed2004

PCR-based screening BAC library and direct end sequencing of BAC clones.

A PCR based strategy was developed which required four steps to identify positive BAC clones from barley BAC (bacterial artificial chromosome) library. In the protocol, two levels of BAC DNA pools (super-pool and pool) were prepared for analysis. One pool is made of one plate DNAs and one super-pool is made of mixing ten consecutive 1/100 diluted pool DNAs (1 approximately 10, 11 approximately 20 ect). First,super-pool DNAs were analysed and then 10 pool DNAs contained in every positive super-pool were analysed. Once positive BAC plates were identified,the bacterial cultures were dipped into PCR mixtures and reaction is made to identify positive BAC clones. The BAC clones identified by each marker were grouped into contigs. BAC-end sequence was obtained from BACs within each contig and primers were designed for the next step chromosome walking. In case of the BAC ends belong to repetitive sequence, the primers were designed based on the subcloned unique band in the contig (identified by Hind III digestion pattern). This method allows us to construct the BAC contig without the costly and time-consuming efforts, and no radioactivity harmful to the body.

Chromosomes, Artificial, Bacterial↗

PubMed2004

Comparative assessment of genotyping methods for study genetic diversity of Fusarium oxysporum isolates.

In this study, we evaluated three PCR-based methods for the molecular typing of nonpathogenic Fusarium oxysporum isolates: random amplified polymorphic DNA (RAPD), polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and amplified fragment length polymorphism (AFLP). The analyses were performed using 64 isolates of F. oxysporum collected from cotton-producing areas in Egypt. A number of polymorphic RAPD, PCR-RFLP and AFLP bands were scored in all isolates and the genetic similarity among them was assessed. Clustering analysis separated the isolates into two main groups, with similarities ranging from 87 to 100% for RAPD, 80 to 100% for PCR-RFLP and 88 to 97% for AFLP, respectively. The obtained data suggested that all three types of markers are equally informative, but the three assays differed in the amount of detected polymorphic bands. AFLP fingerprinting was also found to be more differentiating than other techniques for the typing of F. oxysporum populations.

DNA, Ribosomal Spacer↗

PubMed2005

Rearrangements of IgH, TCRD and TCRG genes as clonality marker of childhood acute lymphoblastic leukemia.

AIM: Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements are excellent patient-specific targets for clonality studies and monitoring of acute lymphoblastic leukemia (ALL). THE AIMS of the study were to select the optimum panel of primers, evaluate incidence of particular types of monoclonal and oligoclonal gene rearrangements and observe alteration of rearrangement profile between diagnosis of ALL and subsequent relapse(s). METHODS: We used polymerase chain reaction (PCR) for amplification of junctional region of rearranged IgH, TCRD and TCRG genes in combination with heteroduplex analysis in polyacrylamide gel. RESULTS: TCRD gene rearrangements were detected in 64%, TCRG - in 45%, and IgH - in 79% of B-precursor ALL patients. For patients with T-ALL, TCRD gene rearrangements were found in 47%, TCRG gene - in 66%, and IgH - in 19% of cases. Evaluation of biallelic and oligoclonal rearrangements was performed in the study. The highest incidence of oligoclonal rearrangements - 25% was shown for IgH gene in patients with B-precursor ALL. Seven pair cases of patients with de novo leukemia and relapses were analyzed and revealed subclonal deviation in rearrangements of IgH or TCR genes during disease evolution. CONCLUSION: We propose a panel of 13 types of rearrangements (primer pairs) sufficient for tumor cell clonality detection in 96% of patients with ALL. Applications of PCR-based analysis of rearranged IgH, TCRD and TCRG genes for discrimination of mono- and oligoclonality and identification of the origin of relapse were demonstrated.

Child↗

PubMed2006

[The relationship between frameshift mutations of transforming growth factor-beta type II receptor, insulin growth factor II receptor, bcl-2 associated X protein, E2F4 and microsatellite instability in gastric carcinoma].

OBJECTIVE: To determine the microsatellite instability in gastric carcinomas, examine the frameshift mutations of transforming growth factor-beta type II receptor (TGFbetaRII), insulin growth factor II receptor (IGFIIR), bcl-2 associated X protein (BAX) and E2F4, and investigate whether or how alterations of the TGFbetaRII, IGFIIR, BAX and E2F4 gene are associated with MSI in gastric cancer. METHODS: Formalin-fixed, paraffin-embedded gastrectomy specimens and matching normal tissues of 65 cases of gastric carcinomas were retrieved from shanghai Ruijin Hospital and Shanghai East Hospital. DNA was extracted from tissue sections using phenol chloral isoamyl alcohol. Sections with no more than 50% of tumor cell areas were isolated by microdissection. DNA was amplified by PCR-based single strand conformation polymorphism (SSCP) for microsatellite analysis and was sequenced directly. Frameshift mutations in the coding regions, repetitive mononucleotide tracts of (A)10 for TGFbetaRII, (G)8 for IGFIIR, (G)8 for BAX, and trinucleotide repeats of (AGC)13 for transcription factors E2F4 were detected using PCR. Tumors were classified as being microsatellite stable (MSS) or having a low frequency of MSI (MSI-L, one of markers different in the tumor) or a high frequency of MSI (MSI-H, two or more of markers different). RESULTS: Eleven cases (18.0%) showed MSI-L, 12 (19.7%) showed MSI-H and 38 (62.3%) cases showed MSS. The mutation rates of TGFbetaRII, IGFIIR, BAX and E2F4 gene were 19.7%, 4.9%, 6.6% and 16.4% respectively. Among the 12 MSI-H gastric cancers, there were 10 TGFbetaRII mutations, 3 IGFIIR mutations, 4 BAX mutations and 10 E2F4 gene mutations. The alterations in the repeats of the related genes presented polymorphisms. Associations of MSI-H status and mutations of the 4 genes were highly significant (P < 0.01, respectively). No repeat tracts mutations in TGFbetaRII, IGFIIR, BAX and E2F4 gene were found in MSS tumors. CONCLUSIONS: The repeat coding regions within TGFbetaRII, IGFIIR, BAX and E2F4 gene are the targets of microsatellite instability. Frameshift mutations of the 4 genes play an important role in the development and progression of gastric cancers with microsatellite instability.

Adult↗

PubMed1995

Semiautomated quantitative detection of loss of heterozygosity in the tumor suppressor gene p53.

The most frequently altered gene in diverse tumor types is the tumor suppressor gene p53. Typically, normal function is inactivated by point mutation of one allele and deletion of the other. Therefore, loss of heterozygosity (LOH) of intragenic polymorphic markers is a strong indication for p53 involvement in a cancerous lesion. This study shows that a highly polymorphic short tandem repeat (STR) within intron 1 of p53 is an excellent marker for quantitative evaluation of LOH in tumor samples, whose multicolor, fluorescently tagged PCR products are analyzed and quantitated on an automated DNA sequencer. The range of error was analyzed in detail. Discrete allelic profiles were obtained following amplification of DNA from microdissected cell samples of patients with urogenital tumors. By calculating qLOH, the relative allele ratio of a tumor compared with healthy tissue, a quantitative expression for the LOH is obtained. PCR-based tumor DNA typing using fluorescent STR primers and automated analysis provides an enhanced level of accuracy and sensitivity required for routine analysis in clinical practice, where large numbers of tumor samples have to be processed.

Alleles↗

PubMed1995

Arab population data on the PCR-based loci: HLA-DQA1, LDLR, GYPA, HBGG, D7S8, Gc, and D1S80.

Allele and genotype frequencies for seven polymerase chain reaction (PCR)-based DNA genetic markers were determined in an Arab sample population. The loci analyzed were HLA-DQA1, LDLR, GYPA, HBGG, D7S8, Gc and D1S80. Results were obtained from the first six loci using the AmpliType HLA-DQ alpha DNA and AmpliType PM PCR Amplification and Typing Kits. The VNTR locus D1S80 PCR product was analyzed by polyacrylamide electrophoresis and silver staining. All loci meet Hardy-Weinberg expectations. The frequency data can be used in forensic analyses and paternity tests to estimate the frequency of a DNA profile in the Arab population.

Alleles↗

PubMed1995

Quantitative non-radioactive clonality analysis of human leukemic cells and progenitors using the human androgen receptor (AR) gene.

Clonal analysis of FACS-purified primitive hematopoietic stem cells and of their progeny as assessed by the progenitors obtained from long-term cultures requires PCR-based approaches, mainly because of the low number of cells available. We have developed a non-radioactive androgen receptor (AR) assay which allows a simple and quantitative evaluation of the clonality of hematopoietic cells and progenitors. In this approach 5' AR primer is labelled by fluorescein and the amplified product is run on a sequencing gel which allows evaluation of the intensity of the fluorescent peaks generated. A computer software then analyzes the reduction of the intensity of the peaks on HpaII-digested samples. In order to determine the feasibility of the technique, we analyzed the clonality of leukemic cells from a patient with an acute-phase CMML which showed a typical clonal pattern of her leukemic DNA sample (WBC = 300 x 10(9)/I) using phosphoglycerate kinase (PGK) analysis. The same sample was then analyzed with either radioactive- or fluorescein-labelled AR primers, showing a typical clonal pattern (complete disappearance of one allele after HpaII digestion). A short-term clonogenic assay was then set up on methylcellulose and clonogenic progenitors were individually analyzed. All 24 colonies tested showed a typical clonal pattern with the disappearance of the same allele on each sample after HpaII digestion, indicating that they all derived from the same leukemic stem cell. Using this approach we then analyzed 94 patients with several hematologic malignancies and quantification of their fluorescent peaks. Fifty-four percent of the patients were clearly heterozygous (ie, a difference of > or = 2 CAG repeats was present between the two copies of the gene) and could be analyzed in an automatic sequencer using the fluorescent primers. Bone marrow mononuclear cells from all patients with acute myeloid leukemia (AML) showed a clonal or oligoclonal pattern at diagnosis whereas a polyclonal pattern was seen when remission was obtained. Similarly, out of 21 patients with a diagnosis of myelodysplastic syndrome (MDS), a clonal pattern was demonstrated in 10 whereas an oligoclonal or non-clonal pattern was shown in 11. These results show that this non-radioactive and safe technology can now be used on a large scale to evaluate the clonality of highly purified hematopoietic stem cells and their progenitors in hematopoietic malignancies and this might allow new insights into the targets of clonal amplification.

Acute Disease↗

PubMed1995

Presentation of a PCR-nuclease protection strategy for minimal residual disease monitoring in B-ALL.

Methods for detecting residual malignant cells in patients suffering from lymphoid malignancies have neither been sufficiently sensitive nor easy to routinize, hampering a potential prediction of disease outcome. Taking advantage of clone-specific DNA sequences, generated during lymphocyte differentiation and the polymerase chain reaction, some strategies have been developed for several groups. Up to now the most specific and sensitive methodology, which consists of designing leukemia-specific oligonucleotides, requires sequencing of the complementary determining region III-DNA for each particular patient and is too laborious to be applied to each case for routine monitoring in most hospital laboratories. In an attempt to achieve an easy way to detect residual malignant cells in B lymphoproliferative diseases, we have used a new PCR-based approach, named here as PCR-nuclease protection assay, consisting of: (i) amplification of DNA segments corresponding to the complementarity determining region III of the immunoglobulin heavy chain genes from samples at disease diagnosis; (ii) isolation of the disease-specific single-stranded DNA; (iii) labeling of the single-stranded DNA to generate specific probes; (iv) hybridization to amplified DNA from samples corresponding to different disease phases; and (v) digestion with S1-nuclease. Using this approach, we could detect one malignant cell in a background of 10(5) healthy cells. The sensitivity and specificity of this approach compares with those of the above mentioned specific oligonucleotide strategy in detecting residual malignant B cells. Moreover, this strategy is much less tedious and could be used by most hospital laboratories.

Base Sequence↗

PubMedDate not supplied

Current molecular methods for plant genome identification.

At least four molecular methods have been used for plant species and plant varietal identification. These include protein isoenzymes, restriction fragment length polymorphisms and more recently two alternate PCR-based procedures. One approach is based broadly on methods which amplify plant DNAs using short oligonucleotide primers to produce complex multibanded 'fingerprint' patterns. The method can be used on any plant, is relatively cheap and easy to set up, but has a number of technical limitations. A second approach is to isolate short tandem repeats (STRs) from a test plant, each example of which is locus-specific and may show length variation between individuals. Results from several STR loci will provide a genetic profile of plant variety. However, in comparison with random amplification methods STRs are costly to establish in both time and resources.

Biotechnology↗

PubMed1995

Allele-specific mutations involved in the pathogenesis of endemic gallbladder carcinoma in Chile.

Although gallbladder carcinoma is one of the most frequent neoplasms in Chile, there is limited information about the molecular changes involved in its pathogenesis. We investigated the incidence of ras gene mutations and loss of heterozygosity (LOH) at the following genes/loci: p53, DCC, rb, 5q 3p, 8p, and 9p. We precisely microdissected 194 relevant areas from paraffin-embedded microslides from 25 gallbladder carcinomas and their accompanying nonneoplastic lesions (which were present in 15 cases) from patients in Chile. The specimens were analyzed by PCR-based assays for LOH, and we designed a RFLP method for ras mutations and immunohistochemistry for p53 protein overexpression. We determined that LOH at p53 (91%), 9p (50%), 8p (44%) and DCC (31%) are frequent events and that LOH at p53, 9p, and DCC are early events, while ras mutations and LOH at 3p, rb, and 5q occurred occasionally. LOH at p53 occurred more frequently and earlier than protein overexpression. The mean number of mutations present in invasive carcinomas was 2.1, and in six cases, LOH at the p53 gene was the sole mutation detected. The same allele was lost in 61 (93%) of 71 nonneoplastic foci as in the corresponding invasive carcinomas for all four mutations studied. The odds of this occurring by chance are approximately 4 x 10(-15). Although clonality cannot be excluded, allelic loss appears to be highly directed, but the mechanism for allele-specific mutations remains to be determined.

Adenocarcinoma↗

PubMed1996

Improved single-step PCR assay for sex identification post-allogeneic sex-mismatched BMT.

A method is presented which allows sex-identification simultaneously of both male and female cells in cell mixtures. The sensitivity of this PCR-based assay is one male cell in 10(6) female cells. The test can be used routinely to analyze bone marrow and peripheral blood lymphocytes post-allogeneic sex-mismatched bone marrow transplantation to detect early engraftment or rejection, as well as host cell regeneration.

Amelogenin↗

PubMed1996

Development of PCR-based tests for the identification of North American isolates of epizootic haemorrhagic disease virus.

A serogroup-specific polymerase chain reaction (PCR) assay and isolate identification strategies (restriction endonuclease analysis (REA) and nucleotide sequencing) were developed for the detection of North American isolates of epizootic haemorrhagic disease virus (EHDV). PCR primers (EHDV-pr4, EHDV-pr5) were designed to hybridize to the L3 gene of a North American isolate of EHDV serotype 1. Total nucleic acid was extracted from preparations of infected tissue culture and PCR was performed using a cDNA-PCR kit, according to the manufacturer's specifications. The PCR assay generated a 459 base pair product from North American isolates of EHDV serotypes 1 and 2, while bluetongue virus (BTV) serotypes 10, 11, 13, and 17, and cell controls, failed to demonstrate PCR products. Slight modifications allowed for the PCR detection of EHDV-1 and -2 in white-tailed deer blood (Odocoileus virginiatus); PCR fragments were not amplified from uninfected deer blood. A number of restriction endonucleases and sequencing primers were evaluated for their utility in isolate identification experiments. Specifically, REA employing HincII and cycle sequencing with an internal primer (EHDV-1-pr3) proved most successful for identifying isolate-specific genome markers. The techniques presented are expected to prove valuable for rapid and specific detection of possible future EHDV incursions in wild and domestic animal species.

Animals↗

PubMed1996

p53 is frequently mutated in Barrett's metaplasia of the intestinal type.

Barrett's Esophagus (BE) is a complication of gastroesophageal reflux in which the normal squamous epithelium of the lower esophagus is replaced by metaplastic tissue. The clinical significance of this condition is the associated predisposition to adenocarcinomas (ADCs). Three types of BE have been characterized: the gastric fundic (F) type, the gastric cardial (C) type, and the intestinal (I) type. The latter is the most closely associated with the development of ADCs; the causes of this bias remain unknown. To determine whether p53 and/or K-ras gene alterations (a) are present in preneoplastic lesions and (b) are associated with a specific histotype, we performed PCR-based denaturing gradient gel electrophoresis (DGGE) analysis of exon 1 (codons 12-13) of K-ras gene and of exons 5-8 of the p53 gene in biopsies obtained from 30 patients with BE of the I type (9 patients), combined I type (I + C +/- F; 10 patients) and non-I type (C, F, or C + F; 11 patients). None of the cases under study revealed K-ras mutations, whereas biopsies from 12 patients showed at least one p53 DGGE variant. Four patients showed the exact same variants in leukocytes also (polymorphisms), whereas eight cases revealed specific DGGE variants only in biopsies. The molecular characterization of these variants revealed that four of them showed a single base pair substitution, and four showed multiple mutations. Of 17 somatic mutations, all but 1 were base pair substitutions located mainly in exons 7 and 8. The majority of these mutations were GC targeted (13 of 16; 81%), 54% (7 of 13) of which were transitions occurring at CpG sites. All somatic mutations were found in BE with at least one I component. The association with the histotype was statistically significant (P < 0.03; pure I type versus non-I type; P < 0.04, combined I type versus non-I type; Fisher's exact test). Loss of heterozygosity in the vicinity of the p53 locus was evaluated by PCR using a highly polymorphic variable number of tandem repeats marker on 25 out of 30 cases. Ninety-two % of the cases analyzed were informative, and none of them showed LOH. In conclusion, we showed that p53 mutations are frequently observed in specimens from BE patients of the I-type, whereas no involvement of K-ras (exon 1) mutational activation was observed. In light of the key roles that the p53 protein plays in controlling cell cycle and cell diploidy, this result may suggest why this type of metaplasia is the most closely associated to the development of ADCs.

Adult↗

PubMed1996

Polymerase chain reaction-based diagnosis of del (5q) in acute myeloid leukemia and myelodysplastic syndrome identifies a minimal deletion interval.

Loss of all or part of the long arm of human chromosome 5 is a recurrent abnormality in patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), especially after chemotherapy for a prior malignancy. It is one of the worst prognostic indicators in AML, associated with chemotherapy resistance and short survival. These deletions center at band 5q31, which has thus been proposed as the location of a tumor suppressor gene; this site is to be distinguished from that observed in 5q- syndrome, centering at 5q32. To define the molecular extent and the clinical prevalence of 5q31 deletions, we collected a series of AML and MDS cases of mixed karyotype, taking care to exclude MDS cases with 5q- syndrome. The samples were analyzed for loss of heterozygosity (LOH) using a panel polymerase chain reaction (PCR)-based microsatellite markers from 5q, comparing malignant cells with normal tissue derived from lymphoblastoid cell lines or buccal mucosa scrapings. Losses were detected in seven of 29 matched samples, including four of 17 with MDS, and three of 12 with AML; six of these seven also had a cytogenetically-visible del(5q) or -5. The one case without a cytogenetic deletion showed molecular loss of three contiguous markers, with retention of flanking markers interleukin-9 (IL-9) and D5S414, and thus contained a small deletion that is below cytogenetic resolution. PCR failed to detect 5q loss in two cases with large cytogenetic deletions, but both had been treated and contained low percentages of malignant cells in the samples. This study thus led to the identification of a case with a minimal deletion for the 5q31 tumor suppressor gene, specified by IL-9-D5S414, that is approximately 1 Mb (2 cM) in extent. Additionally, we demonstrate that PCR-based allelotyping is a reliable method for the detection of chromosomal deletion in myeloid malignancy, providing the specimens contain a high proportion of malignant cells. These studies will help to identify the tumor-suppressor gene at 5q31, and will help to develop molecular methods for diagnosis and monitoring of these disorders.

Acute Disease↗

PubMed1996

Gender determination of forensic samples using PCR amplification of ZFX/ZFY gene sequences.

Determining the gender of an evidentiary sample can be an important part of casework analyses. Gender information, particularly when combined with mitochondrial DNA analysis, can serve to distinguish biological evidence from two people who share the same DNA type(s) but differ by sex. When typing sexual assault evidence, gender information can serve as confirmation that the "sperm fraction" extracted from swabs and stains actually contains male DNA and also as an indicator of the amount of male DNA present in the non-sperm fraction. The PCR-based assay described here relies on amplification of a small, polymorphic region of a homologous zinc finger protein locus present on the X and Y chromosomes. The gender of the sample donor is determined from the PCR product either by Haelll restriction enzyme digestion followed by gel electrophoresis or by hybridization to immobilized sequence specific oligonucleotide probes (reverse dot blot). When using the reverse dot blot approach, amplification and typing of the gender PCR product can be coupled to amplification and typing of the AmpliType HLA DQalpha and PM markers. Sensitivity and mixture studies were performed in addition to the analysis of casework bloodstains and sexual assault kit samples. Additional studies using this gender determination assay are described in the accompanying paper.

Base Sequence↗

PubMed1996

Hybrid survival motor neuron genes in patients with autosomal recessive spinal muscular atrophy: new insights into molecular mechanisms responsible for the disease.

Spinal muscular atrophy (SMA) is a frequent autosomal recessive neurodegenerative disorder leading to weakness and atrophy of voluntary muscles. The survival motor-neuron gene (SMN), a strong candidate for SMA, is present in two highly homologous copies (telSMN and cenSMN) within the SMA region. Only five nucleotide differences within the region between intron 6 and exon 8 distinguish these homologues. Independent of the severity of the disease, 90%-98% of all SMA patients carry homozygous deletions in telSMN, affecting either exon 7 or both exons 7 and 8. We present the molecular analysis of 42 SMA patients who carry homozygous deletions of telSMN exon 7 but not of exon 8. The question arises whether in these cases the telSMN is truncated upstream of exon 8 or whether hybrid SMN genes exist that are composed of centromeric and telomeric sequences. By a simple PCR-based assay we demonstrate that in each case the remaining telSMN exon 8 is part of a hybrid SMN gene. Sequencing of cloned hybrid SMN genes from seven patients, as well as direct sequencing and single-strand conformation analysis of all patients, revealed the same composition in all but two patients: the base-pair differences in introns 6 and 7 and exon 7 are of centromeric origin whereas exon 8 is of telomeric origin. Nonetheless, haplotype analysis with polymorphic multicopy markers, Ag1-CA and C212, localized at the 5' end of the SMN genes suggests different mechanisms of occurrence, unequal rearrangements, and gene conversion involving both copies of the SMN genes. In approximately half of all patients, we identified a consensus haplotype, suggesting a common origin. Interestingly, we identified a putative recombination hot spot represented by recombination-stimulating elements (TGGGG and TGAGGT) in exon 8 that is homologous to the human deletion-hot spot consensus sequence in the immunoglobulin switch region, the alpha-globin cluster, and the polymerase alpha arrest sites. This may explain why independent hybrid SMN genes show identical sequences.

Biomarkers↗

PubMed1998

PCR detection of Y-specific sequences in patients with Ullrich-Turner syndrome: clinical implications and limitations.

Cytogenetic analysis of patients with Ullrich-Turner syndrome (UTS) may fail to detect low levels of Y chromosome mosaicism or Y-derived marker chromosomes. More sensitive polymerase chain reaction (PCR)-based tests have been developed; however, applicability of these data to prognosis of virilization and gonadoblastoma development has not been investigated adequately. We used a multiplex PCR-based method to detect two Y-specific sequences, SRY and AMGLY. Thirteen patients with UTS without cytogenetically detected Y chromosomes were studied. Y-specific sequences were detected in 5 patients by multiplex PCR. A cryptic translocation involving the Y chromosome was found in one patient with severe virilization of external genitalia and a male phenotype. Y chromosomal mosaicism was detected in peripheral blood and in both gonads of one patient, and only in the left gonad of another patient. Existence of a Y-derived marker was demonstrated in 2 patients, one of whom had no testicular tissue or virilization. Consistent with previous reports, we conclude that PCR is more sensitive than classical cytogenetic analysis and detects patients with Y-specific sequences in blood cells. However, the absence of Y-specific material in blood is not a sufficient reason to reject surgical treatment in case of virilization.

Adolescent↗