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PubMed2004

Large BRCA1 gene deletions are found in 3% of German high-risk breast cancer families.

We have tested for large BRCA1 gene rearrangements in German high-risk breast and ovarian cancer families previously screened negative for point mutations by dHPLC and sequencing. Using the novel MLPA method, two deletions of exons 1A, 1B and 2 and exon 17, respectively, were detected in four out of 75 families investigated in Southern Germany. An identical exon 17 deletion with the same breakpoints and a deletion of exons 1A, 1B and 2 were found by fluorescent multiplex PCR in two out of 30 families investigated in Northern Germany. Combining both populations, genomic rearrangements were found in 6% of the mutation-negative families and 3% of all high-risk families and account for 8% of all BRCA1 mutations. Our data indicate that the exon 17 deletion may be a founder mutation in the German population. The prevalence of BRCA1 gene deletions or duplications in our patients is similar to previous reports from Germany and France. Genomic quantification by MLPA is a useful method for molecular diagnostics in high-risk breast cancer families.

Breast Neoplasms↗

PubMed2005

Large genomic rearrangements in MECP2.

In 1999, mutations in the X-linked gene methyl-CpG-binding protein 2 (MECP2) were first reported in patients with Rett syndrome (RTT). The MECP2 gene is located at Xq28 and consists of 4 exons. About 80-90 % of the classic RTT patients harbor mutations in the coding region of MECP2, while the molecular cause is unknown in the remaining 10-20%. Several groups have searched for large rearrangements within the MECP2 and the results indicate that a fraction of MECP2-negative RTT cases has large deletions of the MECP2. In this study we have used the Multiplex Ligation-dependent Probe Amplification (MLPA) technique to screen 45 RTT patients, who have previously been tested negative for mutations in the coding region of MECP2. The MECP2-MLPA is a semi-quantitative multiplex PCR approach. It determines the relative number of copies of each MECP2 exon. With this approach we detected seven RTT patients with genomic deletions and further characterized the deletions using real time quantitative PCR (qPCR) and long-range PCR. The seven patients were given a severity score and their X chromosome inactivation profiles were determined in order to identify a possible genotype-phenotype correlation. The results from this study indicate that large deletions in MECP2 cause classic RTT.

Adolescent↗

PubMed2003

Hereditary nonpolyposis colorectal cancer: frequent occurrence of large genomic deletions in MSH2 and MLH1 genes.

Hereditary nonpolyposis colorectal cancer (HNPCC) is often caused by a deficiency in DNA mismatch repair. By using conventional methods of mutation analysis, point mutations in the DNA mismatch repair genes MSH2 and MLH1 have been detected in up to 64% of patients suspected of HNPCC. However, large genomic deletions cannot be detected by these methods. In our study, we applied a semiquantitative multiplex PCR to detect the proportion of large deletions in patients meeting the Bethesda criteria whose tumours exhibited microsatellite instability (MSI). Of 368 unrelated patients, 180 exhibited MSI. In these patients, 68 disease-causing point mutations (38%) had previously been detected in the MSH2 and MLH1 genes by SSCP, heteroduplex analysis or DHPLC followed by direct sequencing. The remaining 112 patients (including 24 patients with rare missense or other unclarified variants) were examined for large deletions. We identified deletions in 19 patients (10.6%); 11/19 (58%) deletions were located in MSH2 and 8/19 (42%) in MLH1, respectively. The size of deletions ranged from 1 exon to a deletion of a whole gene. Five breakpoints of deletions were sequenced; Alu-repetitive elements were involved in all of them. In patients meeting the Amsterdam criteria the proportion of large deletions was 12.6%. A similar proportion of deletions was found in the group of patients with a positive family history for colorectal cancer and MSI tumours, not meeting the Amsterdam criteria. The results of our study suggest that large genomic deletions in both MSH2 and MLH1 genes play a considerable role in the pathogenesis of HNPCC and should be part of the routine HNPCC mutation detection protocols.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Promoter hypermethylation profile of tumor-associated genes p16, p15, hMLH1, MGMT and E-cadherin in oral squamous cell carcinoma.

Aberrant promoter hypermethylation of tumor-associated genes leading to their inactivation is a common event in many cancer types. Using a sensitive restriction-multiplex PCR method, we studied the promoter hypermethylation profile of the p16, p15, hMLH1, MGMT and E-cad genes in oral squamous cell carcinoma (OSCC) of Indians. We analyzed a total of 51 samples for the p15 tumor-suppressor gene and 99 samples for each of the remaining genes. Our studies indicate an incidence of promoter hypermethylation of 23% each for p16 and p15, 8% for hMLH1, 41% for MGMT and 35% for E-cad. We observed aberrant hypermethylation of the promoter region of at least 1 of these genes in 74.5% of cases (n = 51) for which all the 5 genes were studied. Abnormal methylation was detected in tumors irrespective of stage and location in the oral cavity, whereas no abnormal methylation was detectable in normal oral squamous tissues obtained from 25 OSCC patients. Detection of aberrant hypermethylation patterns of cancer-associated genes listed above is therefore suitable for diagnosis of OSCC in individuals at high risk for this disease.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Genomic gains on chromosome 1q in retinoblastoma: consequences on gene expression and association with clinical manifestation.

Many retinoblastomas (Rbs) show genomic alterations in addition to mutational loss of both normal RB1 alleles. The most frequent of these changes are gains on chromosomes 1q and 6p and losses on 16q. To identify the genes targeted by gains on chromosome 1q, we used quantitative-multiplex PCR to determine DNA copy number changes in 76 primary tumors and 6 Rb cell lines. In addition, in 21 of these tumors, gene expression was analyzed by cDNA microarray hybridization. Increased copy numbers of loci on chromosome 1q were present in 34 (45%) primary tumors and in all 6 cell lines. Two regions of gain emerged, one in 1q32 and another in 1q21. Tumors with 1q gains showed higher RNA expression of several genes in these 2 regions. The clinical manifestation of tumors with and without gains was similar with regard to many aspects, including size, necrosis and calcification. However, the distribution of age at diagnosis was remarkably distinct, with earlier diagnosis in tumors without gains. This suggests that these tumors either are initiated earlier or grow faster than tumors with gains. This association with clinical manifestation indicates that gains on 1q are significant for the biology of Rb. The genes on 1q with copy number gains and overexpression are candidates that need to be tested for their individual contribution to the progression of Rb.

Adolescent↗

PubMed2005

Type V collagen regulates the expression of apoptotic and stress response genes by breast cancer cells.

Type V collagen is a "minor" component of normal human breast stroma, which is subjected to over-deposition in cases of ductal infiltrating carcinoma (DIC). We reported that, if used as a culture substrate for the DIC cell line 8701-BC, it exhibited poorly-adhesive properties and restrained the proliferative and motile behavior of the cell subpopulation able to attach onto it. Moreover, this collagen species was able to trigger DNA fragmentation and impair survival of 8701-BC cells. In this study, we have extended our investigation with the aim to obtain further evidence that the death induced by type V collagen was of the apoptotic type by (i) microscopic detection and quantitation of Apoptag-labeled cells, (ii) analysis of the expression levels of selected genes coding for apoptosis-linked factors, caspases, and stress-response proteins by conventional and semi-quantitative multiplex PCR, and (iii) evaluation of the extent of caspase activation by chromogenic assay. We report here that type V collagen is able to determine an increase in the percentage of Apoptag-positive cells, to up-regulate Bcl-xS, Bad, Dap kinase, hsf-1, mthsp75, caspase-1, -5, -8, -9, and -14, whilst down-regulating Bcl-2, Bcl-xbeta, and hsp60. Treatment of cell lysates with chromogenic tetrapeptide substrates specific for caspase-1, -5, -8, and -9 demonstrated a marked increase of enzymatic activity in the presence of type V collagen. Our data validate 8701-BC cell line as a suitable "in vitro" model for further and more detailed studies on the molecular mechanisms of the death response induced by type V collagen on primary DIC cells.

Apoptosis↗

PubMed2002

HPV16 and HPV18 in genital tumors: Significantly different levels of viral integration and correlation to tumor invasiveness.

The integration of the high-risk HPV16 and HPV18 types into the cell genome is considered an important step in malignant transformation. The relationship between the physical status of the virus and clinical/pathological parameters was studied by type-specific and multiplex PCR for E6, E2, and E1 sequences in 86 genital tumors from different sites, consisting of 69 invasive carcinomas (including 5 microinvasive carcinomas), 9 carcinomas in situ, 6 severe dysplasias, and 2 moderate dysplasias. Forty tumors contained HPV16 (46.6%), 7 HPV18 (8.1%), and 39 both viruses (45.3%). HPV16 DNA was found either as pure integrant (35.4%), or pure episome (36.7%), or a mixture of both (27.8%). Conversely, all 46 lesions containing HPV18 showed pure integrated forms. The physical status of both types was not related to the tumor site, the tumor/node/metastasis stage, or the histological differentiation grade of the invasive carcinomas. HPV16 integration was significantly associated with invasiveness. Interestingly, in double infections when HPV16 coexisted with HPV18, its genome was found more frequently in episomal form than in single infections where, conversely, it was mostly integrated (P < 0.0001), suggesting a sort of competition for cell integration sites. The complete HPV18 integration, even in pre-neoplastic lesions, indicates a different behavior in genital transformation compared with HPV16 and may reflect a major aggressiveness of this viral type. In conclusion, virus typing in conjunction with the evaluation of the integration status may provide a better prognostic evaluation together with an improved diagnosis.

Adult↗

PubMed2004

Genetic variability of human rotavirus strains isolated from Eastern and Northern India.

An epidemiological study was conducted in Eastern and Northern India to determine the genomic diversity of rotaviruses in these parts of the country. In 2001, a total of 126 Group A rotavirus positive samples were detected from children below 4 years of age with diarrhoea from Kolkata, Dibrugarh and Bhubaneswar in Eastern India, and Chandigarh, a city in Northern India. All the samples were genotyped for VP7 (G-type) and VP4 (P-type) gene by reverse transcription (RT) and multiplex PCR using different type specific primers. The strains with G1P[8] (32.5%) was predominant as reported earlier [Das et al. (2002) J Clin Microbiol 40:146-149] followed by G2P[4](4.7%) and only one sample was of G4P[8] specificity. Along with these common types some rare strains like G1P[6], G2P[8], G2P[6], G4P[4], and G4P[6] were also detected in 14.3% of cases. Thirty percent of samples in this study were mixed infections and 21 (16.7%) specimens remained untypeable either for the VP7 or for the VP4 gene. After sequencing of the VP7 gene, two G9 strains (RMC321 and ISO-3) were identified with P[8] and P[19] specificities. Sequence analysis revealed that they have much lower homology to the G9 strains (116E, INL1, and G16) isolated earlier from Indian subcontinent, but have much higher homology to isolates from Argentina, Brazil, Malawi, Taiwan, and USA suggesting a separate progenitor for these strains.

Amino Acid Sequence↗

PubMed1993

High-risk genital papillomaviruses and degree of dysplastic changes in the cervix: a prospective study by fast multiplex polymerase chain reaction in Belgium.

The epidemiologic studies with the least selection bias do not support the hypothesis that HPV types 16 and/or 18 are strongly associated with cervical cancer. In this preliminary report, we describe our findings regarding type 16, 18, and 33 detection rates in 323 normal and 71 dysplastic or neoplastic cervical scrapes using fast multiplex PCR. This modified PCR technique has been shown to be the most sensitive, specific, and reproducible DNA detection method for large epidemiologic studies. The results indicate a high relative risk of increasingly severe cervical abnormality associated with the presence of high-risk HPV DNA. The analysis of the prevalence and age data according to CIN status by non-parametric statistic tests highlights the importance of other factors inversely correlated with age in the cervical transformation process.

Adult↗

PubMed2004

Antigenic and molecular analysis of influenza A (H3N2) virus strains isolated from a localised influenza outbreak in South Africa in 2003.

A severe acute institutional influenza outbreak occurred in a police residential college in Pretoria amongst new recruits and staff members at the end of May 2003. The outbreak was characterised by marked illness which affected a total of 648 students, 26 of whom were admitted to hospital. Symptoms included pyrexia, severe headache, and myalgia. The attack rate per dormitory building ranged from 20 to 47%, with an overall attack rate of 34%. Throat swabs and bronchoalveolar lavage specimens were sent to the National Institute for Communicable Diseases (NICD) from 20 patients. All were positive for influenza A by multiplex PCR and/or indirect immunofluorescence, and were further identified as subtype H3N2. Additional specimens from sporadic influenza cases in Johannesburg and surrounding areas were collected through the NICD active viral surveillance programme for respiratory viral testing and were also positive for influenza A H3N2 viruses. Viruses isolated from patients from both the institutional outbreak as well as from sporadic cases were analysed both antigenically and at the molecular level to determine the characteristics of the influenza strain responsible for the epidemic. The results showed clearly that the outbreak was caused by the introduction in 2003 into South Africa of the novel A/Fujian/411/02-like H3N2 influenza strain, which is antigenically distinct from the A/Panama/2007/99 vaccine strain. The rapid spread of these variant viruses to the southern hemisphere indicates that the H3N2 component of the influenza vaccine needs to be updated for the 2004 southern hemisphere winter.

Antigens, Viral↗

PubMed2006

Detection of specific human papillomavirus types in paraffin-embedded sections of cervical carcinomas.

Human papillomaviruses (HPV) are the causative agents of most cervical carcinomas. A complete understanding of the HPV types that cause cervical carcinoma is needed as vaccines are designed. Fresh tissues are not always available for such studies. We therefore sought to determine the feasibility of HPV studies using formalin-fixed, paraffin-embedded sections of 56 cervical carcinomas, correlating typing information with the pathology and physical state of the HPV sequences within cells. Sections from each specimen were used to extract and purify DNA. Specific HPV types were identified using a PCR/reverse blot strip assay. Tyramide signal-amplified, fluorescent DNA in situ hybridization (FISH) was used to localize HPV within cells. Human beta-globin sequences were amplified in DNA from all specimens. HPV sequences from oncogenic types were identified in 52 of 56 (92.9%) by PCR/reverse blot strip assay, and in one additional case using an HPV 16 multiplex PCR assay. HPV 16 was the most commonly detected type, present in most cases as a solitary isolate. Thirty- five of 42 HPV 16 or HPV 18 PCR-positive specimens were also positive in the FISH assay, in most cases in a pattern consistent with viral integration. We conclude that HPV typing from formalin-fixed, paraffin-embedded sections of cervical carcinomas is possible, with a sensitivity that is similar to that found in studies using fresh tissue.

Animals↗

PubMed2006

Correlates of placental infection with cytomegalovirus, parvovirus B19 or human herpes virus 7.

Vertical transmission of viruses is an important cause of morbidity in the fetus and neonate. Placental viral infection indicates risk of vertical transmission, but not always transmission to, or disease of the fetus. Specimens from mothers and babies from three groups-two prospective and one retrospective cohort-were tested for pathogens of teratogenic potential using multiplex PCR. Placental infection was present in 13% of the 105 samples collected. Assessment of the prospective cohorts showed cytomegalovirus (CMV) detected in 4% of placentae from unselected women, parvovirus B19 in 1% and Ureaplasma parvum in 1% of placentae. In a retrospective cohort of women at high risk of transmitting congenital infection due to seroconversion during pregnancy, miscarriage or stillbirth, CMV was detected in 64% and human herpes virus type 7 in 9% of placentae. Of 14 PCR-positive placentae, two were associated with the birth of a living symptomatic infant, two with stillbirth, one with miscarriage, and two with elective terminations of pregnancy. Directed laboratory assessment of women at high risk of transmitting congenital infection, on the basis of clinical or laboratory markers, is important for accurate diagnosis of adverse outcomes of pregnancy. However, routine screening for viruses in the placentae from women with a low-risk serological profile for transmitting congenital infection is unlikely to result in significant numbers of additional diagnoses and is confounded by inadequacy of current diagnostic methods. The major pathogen detected in all cases of placental infection associated with fetal death was human CMV.

Adolescent↗

PubMed2006

Outbreak of sapovirus infection among infants and children with acute gastroenteritis in Osaka City, Japan during 2004-2005.

One hundred and twenty five fecal specimens were collected from sporadic cases of acute gastroenteritis in a pediatric clinic in Osaka City, Japan from July 2004 to June 2005 and tested for the presence of rotavirus, norovirus, sapovirus, astrovirus, and adenovirus by RT-multiplex PCR. Among diarrheal viruses detected, norovirus was the most prevalent (19.2%, 24 of 125), followed by group A rotavirus (18.4%, 23 of 125), astrovirus (1.6%, 2 of 125), and adenovirus (0.8%, 1 of 125), respectively. Interestingly, sapovirus infection was identified with high incidence of 17.6% (22 of 125). Sapovirus was subjected to molecular genetic analysis by sequencing. It was found that sapovirus detected in this study was classified into four genotypes (GI/1, GI/4, GI/6, and GI/8), and sapovirus GI/6 was predominant, followed by GI/8 and accounted for 81.8% (18 of 22) and 9.1% (2 of 22), respectively. It was noteworthy that sapovirus GI/6 infection was apparently confined within the period of 5 months (August 2004 through December 2004). This pattern of infection implied the outbreak of sapovirus GI/6 in these patients, which was the first outbreak of acute gastroenteritis attributed to sapovirus in Osaka City. Another interesting feature of the study was the appearance of two novel, naturally occurring recombinant sapoviruses circulating in the Japanese population studied. This report confirmed the presence as well as the importance of sapovirus causing acute gastroenteritis among Japanese infants and children.

Aging↗

PubMed2025

Clinical and Epidemiological Insights into a Parainfluenza Virus Type 3 Outbreak in Multiple Myeloma Patients.

Human parainfluenza virus type 3 (HPIV-3) can be responsible for mild to severe respiratory infections and hospital epidemics. We investigated an outbreak in a hematology unit. Respiratory viruses were screened using multiplex PCR. HPIV-3 quantification and whole-genome sequencing were performed on HPIV-3 positive respiratory samples. Clinical characteristics, infection progression, incidence rates of respiratory viruses within the hospital and detection of respiratory viruses were documented, along with the reinforcement of infection prevention and control (IPC) measures implemented. Between November 2022, and January 2023, HPIV-3 was identified in 20 of 113 hematology patients (17.7%), of whom 80% had multiple myeloma. A majority of HPIV-3-positive patients developed pneumonia (60%), and mortality was notably higher (35%) compared to patients who were negative (3%, p < 0.0001). Respiratory HPIV-3 viral loads were similar between patients with and without pneumonia. In parallel, HPIV-3 incidence in the hospital overall was lower than in the hematology unit (p < 0.0001). Air virus screening showed the detection of HPIV-3 in the air in different areas, and whole-genome sequencing confirmed the circulation of a single HPIV-3 strain. Strengthened IPC measures were associated with the containment of the outbreak. HPIV-3 has high epidemic potential in patients with multiple myeloma and causes severe infections. Our findings highlight the need for routine HPIV-3 testing in hematology units.

Humans↗

PubMed2007

Expression patterns of ubiquitin, heat shock protein 70, alpha-actin and beta-actin over the molt cycle in the abdominal muscle of marine shrimp Litopenaeus vannamei.

Crustacean muscle growth is discontinuous due to molt cycle. To characterize molt-related gene expression patterns, we studied the mRNA levels of molecular chaperone-ubiquitin and heat shock protein 70 (Hsp 70) in comparison with muscle protein alpha-actin and beta-actin in marine shrimp Litopenaeus vannamei. Total RNA from abdominal muscle was isolated from 3-month-old animals in six different molt stages. The mRNA levels of target genes were detected by reverse-transcriptase-multiplex PCR and expressed as the ratio to elongation factor-1alpha. Ubiquitin mRNA levels were relatively steady over all stages of the molt cycle. Hsp70 levels were not detectable in early postmolt and late premolt stages, but showed a progressive increase from late postmolt to intermolt stages. Expression levels of alpha-actin gene were lower during postmolt, reached a plateau in intermolt and remained relatively high in premolt stage. Levels of beta-actin increased progressively from postmolt to intermolt, reaching a maximum value in premolt. Therefore, the mRNAs encoding for ubiquitin and Hsp 70 in abdominal muscle did not increase significantly in premolt stages, which is typically associated with claw muscle degradation. Muscle structural alpha-actin and cytoskeletal beta-actin were increased during intermolt and premolt stages, suggesting high muscle growth during these stages in the abdominal muscle of the L. vannamei.

Abdominal Muscles↗

PubMed2004

Increased risk for aplastic anemia and myelodysplastic syndrome in individuals lacking glutathione S-transferase genes.

BACKGROUND: Aplastic anemia (AA) and myelodysplastic syndrome (MDS) are marrow failure states that may be associated with chromosomal instability. An absence of the glutathione S-transferase (GST) enzyme may genetically predispose individuals to AA or MDS. PROCEDURE AND RESULTS: To test this hypothesis, we determined the GSTM1 and GSTT1 genotypes in a total of 196 patients using multiplex PCR. The GSTT1 null genotype was found to be overrepresented in Caucasian, Asian, and Hispanic patients with either AA or MDS. We confirmed a difference in the expected frequency of the GSTM1 null genotype in Caucasian MDS patients. The double null GSTM1/GSTT1 genotype was also overrepresented in Caucasian AA and MDS patients. In our population, 26% of AA patients and 40% of MDS patients had a chromosomal abnormality identified by karyotype or FISH analyses for chromosomes 7 and 8. Patients with AA and the GSTT1 null genotype had an increased frequency of chromosomal abnormalities (P = 0.003). CONCLUSION: There seems to be an increased risk for AA and MDS in individuals lacking GSTT1 or both GSTM1/GSTT1.

Adolescent↗

PubMed2004

Prenatal diagnosis for arginase deficiency by second-trimester fetal erythrocyte arginase assay and first-trimester ARG1 mutation analysis.

Hyperargininemia is a progressive neurometabolic disorder caused by deficiency of hepatic cytosolic arginase I, resulting from mutations in the ARG1 gene. We diagnosed arginase deficiency in a three-year-old male child of first-cousin Palestinian Arab parents. Prenatal diagnosis of an unaffected fetus was achieved in the second trimester of a subsequent pregnancy by cordocentesis and analysis of arginase activity in fetal erythrocytes. ARG1 mutation analysis in the proband revealed homozygosity for a deletion of 10,753 bp extending from the first intron to beyond the poly (A) site of the gene. This is the first gross deletion in the ARG1 gene to be identified and the first mutation to be described in an arginase-deficient patient of this ethnic origin. The identification of the ARG1 deletion in this family enabled first-trimester prenatal diagnosis in a subsequent pregnancy by multiplex PCR analysis performed on chorionic villous DNA.

Adult↗

PubMed2004

Prenatal diagnosis of DMD in a female foetus affected by Turner syndrome.

OBJECTIVES: We report on a prenatal diagnosis of DMD complicated by a 45,X karyotype that was revealed only in the chorionic villus long-term culture. METHODS: Cytogenetic investigations were performed on both short-term (STC) and long-term cultures (LTC) of the chorionic villus sample. Familial segregation was performed using a panel of intragenic polymorphic markers, and multiplex PCR was used to characterize exonic deletion. RESULTS: Investigations performed for sex determination after STC of the chorionic villus sample showed a normal karyotype 46,XX, while the karyotype performed after LTC revealed a homogeneous monosomy X. Cytogenetic analysis performed on amniotic fluid cells showed 45,X/46,XX mosaicism. Familial segregation analysis for DMD showed loss of heterozygosity for the STR49 marker in the DNA of the proband, her mother and the foetus. Dystrophin gene analysis on the 45,X cells led to the identification of a deletion of exon 50. CONCLUSIONS: The report described a rare situation of monosomy X associated with a DMD genotype. The data confirmed the DMD carrier status of the proband and her mother and indicated that the foetus had a high risk to combine a Turner phenotype and DMD. This study illustrated the potential risk of using short-term culture of villi as the only source of biological material for prenatal diagnosis.

Diagnosis, Differential↗