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1.  An Improved RSP Method to Detect HpaI Polymorphism in the Apolipoprotein C-1 Gene Promoter 
BMC Medical Genetics  2002;3:13.
Background
An apolipoprotein C1 gene promoter polymorphism (CGTT insertion at position -317) is associated with familial dysbetalipoprotemia, cardiovascular diseases, and Alzheimer's disease. Restriction site polymorphism (RSP) assays were previously established to detect this polymorphism. In this study, we introduce an improved RSP assay to detect this polymorphism.
Methods
This method included newly designed primers and only one round of PCR amplification which yields one short and specific APOC1 fragment followed by HpaI digestion. Briefly, It consists of three steps: 1) one round of PCR amplification of DNA sample, 2) HpaI enzyme digestion of PCR products, and 3) electrophoresis on an agarose gel to visualize the genotypes. This improved RSP method was applied to genotype 92 human samples collected from The Johns Hopkins Hospital.
Results
The observed allele frequencies for H1 and H2 from 92 genotyped human subjects were 0.707 and 0.293 respectively. The H2 allele frequency in the black subjects (0.350) was significantly (p = 0.024) higher than that in the white subjects (0.177). This method was more economical and convenient than the methods previously reported to detect this mutation in the APOC1 gene.
Conclusions
This assay will be readily applied to screen large sample sizes for population studies in a simple and cost effective way.
doi:10.1186/1471-2350-3-13
PMCID: PMC137584  PMID: 12429068
2.  Gene expression patterns vary in clonal cell cultures from Rett syndrome females with eight different MECP2 mutations 
BMC Medical Genetics  2002;3:12.
Background
Females with the neurological disorder Rett syndrome are heterozygous for mutations in X-linked MECP2 that encodes methyl-CpG binding protein 2 (MeCP2) thought to act as a transcriptional repressor. To identify target genes for MeCP2 modulation, we studied global gene expression in single cell-derived wild-type and mutant MECP2 expressing fibroblast clones with four common mutations (R106W, R306C, 705delG, 1155del32) and in lymphoblastoid cell lines (LCLs) that included four mutant MeCP2 (T158M, 803delG, R168X and 1159del28) expressing, and five (1159del28, R106W, R255X, 803delG, 803delG) wild-type MeCP2 expressing lines.
Methods
Clonality and mutation status were verified by androgen receptor methylation assays for X-inactivation and by sequencing MECP2 transcripts. Expression studies were done with oligonucleotide microarrays (Affymetrix U95) and verified with real-time quantitative RT-PCR using Sybr Green.
Results
Expression of 49 transcripts was increased, and expression of 21 transcripts was decreased, in at least 3 of 4 mutant/wild-type fibroblast comparisons. Transcript levels of 11 genes, determined by quantitative RT-PCR, were highly correlated with the microarray data. Therefore, multiple additional clones from two Rett individuals were tested by RT-PCR only. Striking expression differences were found in both mutant and wildtype MeCP2 expressing clones. Comparing expression profiles of lymphoblastoid cell lines yielded 16 differentially expressed genes.
Conclusions
MeCP2 deficiency does not lead to global deregulation of gene expression. Either MeCP2's in vivo function does not involve widespread transcriptional repression, or its function is redundant in cell types that also express other methyl-CpG binding proteins. Our data suggest that clonal fibroblast strains may show substantial inter-strain variation, making them a difficult and unstable resource for genome-wide expression profiling studies.
doi:10.1186/1471-2350-3-12
PMCID: PMC137585  PMID: 12418965
3.  Telomerase activity in human leukemic cells with or without monosomy 7 or 7q- 
BMC Medical Genetics  2002;3:11.
Background
In bone marrow material from patients with various leukemias we noted that samples with either a deletion on the long arm of one chromosome 7 (7q-) or a monosomy 7 had a higher telomerase activity. Considering that introduction of a chromosome 7 into a cancer cell line had been reported to eliminate telomerase activity, that 7q- is a common negative prognostic finding in cancers, and that the deleted segment (band 7q31) contains an unidentified tumor suppressor gene, we wondered if this gene might be a telomerase inhibitor.
Results
We found no significant difference in telomerase activity between the three groups of patient samples. In contrast to reports on tumor cell lines we observed no amplification of the telomerase genes.
Methods
We analyzed telomerase activity and copy number of the telomerase genes hTERT and hTR in frozen archival bone marrow samples from leukemia patients with a referral diagnosis of AML, and either a monosomy for chromosome 7, a deletion on the long arm of chromosome 7 (7q-), or none of these aberrations. Telomerase activity was measured with a commercially available kit, and the copy number of the telomerase genes was tested by FISH.
Conclusions
We found no evidence of a telomerase inhibitor in band 7q31. The lack of telomerase gene amplification found in cell lines from solid tumors could reflect that this amplification is a property of solid tumors, not of hematological cancers.
doi:10.1186/1471-2350-3-11
PMCID: PMC137583  PMID: 12423555
4.  Patients affected with Fabry disease have an increased incidence of progressive hearing loss and sudden deafness: an investigation of twenty-two hemizygous male patients 
BMC Medical Genetics  2002;3:10.
Background
Fabry disease (FD, OMIM 301500) is an X-linked inborn error of glycosphingolipid metabolism due to the deficient activity of alpha-galactosidase A, a lysosomal enzyme. While the progressive systemic deposition of uncleaved glycosphingolipids throughout the body is known to have protean clinical manifestations, few data are available regarding the cochlear involvement.
Methods
We non-invasively investigated cochlear functions in 22 consecutive hemizygous males (age 19–64 years, mean 39) affected with classic FD. Conventional audiometry, tympanometry, ABR audiometry, otoacoustic emissions were performed in all patients, together with medical history record and physical examination as part of an exhaustive baseline evaluation prior to enzyme replacement therapy.
Results
A total of 12 patients (54.5%) with classic FD were found to have abnormal audition. Five patients had progressive hearing loss and seven patients (32%) experienced sudden deafness. In addition, a hearing loss on high-tone frequencies was found in 7 out of the 10 remaining patients without clinical impairment, despite their young age at time of examination. The incidence of hearing loss appeared significantly increased in FD patients with kidney failure (P < 0.01) or cerebrovascular lesions (P < 0.01), whereas there was no correlation with left ventricular hypertrophy. In addition, tinnitus aurium was also found in six patients (27%).
Conclusion
This is the first evidence of a high incidence of both progressive hearing loss and sudden deafness in a cohort of male patients affected with classic Fabry disease. The exact pathophysiologic mechanism(s) of the cochlear involvement deserves further studies.
doi:10.1186/1471-2350-3-10
PMCID: PMC134464  PMID: 12377100
5.  HLA-A and -B alleles and haplotypes in hemochromatosis probands with HFE C282Y homozygosity in central Alabama 
Background
We wanted to quantify HLA-A and -B allele and haplotype frequencies in Alabama hemochromatosis probands with HFE C282Y homozygosity and controls, and to compare results to those in other populations.
Methods
Alleles were detected using DNA-based typing (probands) and microlymphocytotoxicity (controls).
Results
Alleles were determined in 139 probands (1,321 controls) and haplotypes in 118 probands (605 controls). In probands, A*03 positivity was 0.7482 (0.2739 controls; p =< 0.0001; odds ratio (OR) 7.9); positivity for B*07, B*14, and B*56 was also increased. In probands, haplotypes A*03-B*07 and A*03-B*14 were more frequent (p < 0.0001, respectively; OR = 12.3 and 11.1, respectively). The haplotypes A*01-B*60, A*02-B*39, A*02-B*62, A*03-B*13, A*03-B*15, A*03-B*27, A*03-B*35, A*03-B*44, A*03-B*47, and A*03-B*57 were also significantly more frequent in probands. 37.3% of probands were HLA-haploidentical with other proband(s).
Conclusions
A*03 and A*03-B*07 frequencies are increased in Alabama probands, as in other hemochromatosis cohorts. Increased absolute frequencies of A*03-B*35 have been reported only in the present Alabama probands and in hemochromatosis patients in Italy. Increased absolute frequencies of A*01-B*60, A*02-B*39, A*02-B*62, A*03-B*13, A*03-B*15, A*03-B*27, A*03-B*44, A*03-B*47, and A*03-B*57 in hemochromatosis cohorts have not been reported previously.
doi:10.1186/1471-2350-3-9
PMCID: PMC137582  PMID: 12370085
allele; ancestral haplotype; haplotype; HFE; HLA; hemochromatosis; iron; iron overload; population genetics
6.  In vitro correction of cystic fibrosis epithelial cell lines by small fragment homologous replacement (SFHR) technique 
Background
SFHR (small fragment homologous replacement)-mediated targeting is a process that has been used to correct specific mutations in mammalian cells. This process involves both chemical and cellular factors that are not yet defined. To evaluate potential of this technique for gene therapy it is necessary to characterize gene transfer efficacy in terms of the transfection vehicle, the genetic target, and the cellular processing of the DNA and DNA-vehicle complex.
Methods
In this study, small fragments of genomic cystic fibrosis (CF) transmembrane conductance regulator (CFTR) DNA, that comprise the wild-type and ΔF508 sequences, were transfected into immortalized CF and normal airway epithelial cells, respectively. Homologous replacement was evaluated using PCR and sequence-based analyses of cellular DNA and RNA. Individual stages of cationic lipid-facilitated SFHR in cultured cell lines were also examined using transmission electron microscopy (TEM).
Results
We demonstrated that the lipid/DNA (+/-) ratio influences the mode of entry into the cell and therefore affects the efficacy of SFHR-mediated gene targeting. Lipid/DNA complexes with more negative ratios entered the cell via a plasma membrane fusion pathway. Transfer of the DNA that relies on an endocytic pathway appeared more effective at mediating SFHR. In addition, it was also clear that there is a correlation between the specific cell line transfected and the optimal lipid/DNA ratio.
Conclusions
These studies provide new insights into factors that underlie SFHR-mediated gene targeting efficacy and into the parameters that can be modulated for its optimization.
doi:10.1186/1471-2350-3-8
PMCID: PMC130050  PMID: 12243649
gene therapy; cystic fibrosis transmembrane conductance regulator (CFTR); gene targeting; transmission electron microscopy (TEM); transfection
7.  Search for intracranial aneurysm susceptibility gene(s) using Finnish families 
Background
Cerebrovascular disease is the third leading cause of death in the United States, and about one-fourth of cerebrovascular deaths are attributed to ruptured intracranial aneurysms (IA). Epidemiological evidence suggests that IAs cluster in families, and are therefore probably genetic. Identification of individuals at risk for developing IAs by genetic tests will allow concentration of diagnostic imaging on high-risk individuals. We used model-free linkage analysis based on allele sharing with a two-stage design for a genome-wide scan to identify chromosomal regions that may harbor IA loci.
Methods
We previously estimated sibling relative risk in the Finnish population at between 9 and 16, and proceeded with a genome-wide scan for loci predisposing to IA. In 85 Finnish families with two or more affected members, 48 affected sibling pairs (ASPs) were available for our genetic study. Power calculations indicated that 48 ASPs were adequate to identify chromosomal regions likely to harbor predisposing genes and that a liberal stage I lod score threshold of 0.8 provided a reasonable balance between detection of false positive regions and failure to detect real loci with moderate effect.
Results
Seven chromosomal regions exceeded the stage I lod score threshold of 0.8 and five exceeded 1.0. The most significant region, on chromosome 19q, had a maximum multipoint lod score (MLS) of 2.6.
Conclusions
Our study provides evidence for the locations of genes predisposing to IA. Further studies are necessary to elucidate the genes and their role in the pathophysiology of IA, and to design genetic tests.
doi:10.1186/1471-2350-3-7
PMCID: PMC119849  PMID: 12153705
8.  SNP analysis of the inter-alpha-trypsin inhibitor family heavy chain-related protein (IHRP) gene by a fluorescence-adapted SSCP method 
Background
Single-nucleotide polymorphisms (SNPs) are considered to be useful polymorphic markers for genetic studies of polygenic traits. Single-stranded conformational polymorphism (SSCP) analysis has been widely applied to detect SNPs, including point mutations in cancer and congenital diseases. In this study, we describe an application of the fluorescent labeling of PCR fragments using a fluorescent-adapted primer for SSCP analysis as a novel method.
Methods
Single-nucleotide polymorphisms (SNPs) of the inter-alpha-trypsin inhibitor family heavy chain-related protein (IHRP) gene were analyzed using a fluorescence-adapted SSCP method. The method was constructed from two procedures: 1) a fluorescent labeling reaction of PCR fragments using fluorescence-adapted primers in a single tube, and 2) electrophoresis on a non-denaturing polyacrylamide gel.
Results
This method was more economical and convenient than the single-stranded conformational polymorphism (SSCP) methods previously reported in the detection of the labeled fragments obtained. In this study, eight SNPs of the IHRP gene were detected by the fluorescence-adapted SSCP. One of the SNPs was a new SNP resulting in an amino acid substitution, while the other SNPs have already been reported in the public databases. Six SNPs of the IHRP were associated with two haplotypes.
Conclusions
The fluorescence-adapted SSCP was useful for detecting and genotyping SNPs.
doi:10.1186/1471-2350-3-6
PMCID: PMC117798  PMID: 12147176
9.  Mutational analysis of Peroxiredoxin IV: exclusion of a positional candidate for multinodular goitre 
Background
Multinodular goitre (MNG) is a common disorder characterised by an enlargement of the thyroid, occurring as a compensatory response to hormonogenesis impairment. The incidence of MNG is dependent on sex (female:male ratio 5:1) and several reports have documented a genetic basis for the disease. Last year we mapped a MNG locus to chromosome Xp22 in a region containing the peroxiredoxin IV (Prx-IV) gene. Since Prx-IV is involved in the removal of H2O2 in thyroid cells, we hypothesize that mutations in Prx-IV gene are involved in pathogenesis of MNG.
Methods
Four individuals (2 affected, 2 unrelated unaffected) were sequenced using automated methods. All individuals were originated from the original three-generation Italian family described in previous studies. A Southern blot analysis using a Prx-IV full-length cDNA as a probe was performed in order to exclude genomic rearrangements and/or intronic mutations. In addition a RT-PCR of PRX-IV was performed in order to investigate expression alterations.
Results
No causative mutations were found. Two adjacent nucleotide substitutions were detected within introns 1 and 4. These changes were also detected in unaffected individuals, suggesting that they were innocuous polymorphisms. No gross genomic rearrangements and/or restriction fragment alterations were observed on Southern analysis. Finally, using RT-PCR from tissue-specific RNA, no differences of PRX-IV expression-levels were detected between affected and unaffected samples.
Conclusions
Based on sequence and genomic analysis, Prx-IV is very unlikely to be the MNG2 gene.
PMCID: PMC117784  PMID: 12135533
10.  A PCR-mutagenesis strategy for rapid detection of mutations in codon 634 of the ret proto-oncogene related to MEN 2A. 
Background
Multiple endocrine neoplasias type 2A (MEN 2A) is a dominantly inherited cancer syndrome. Missence mutations in the codon encoding cysteine 634 of the ret proto-oncogene have been found in 85% of the MEN 2A families. The main tumour type always present in MEN 2A is medullar thyroid carcinoma (MTC). Only 25% of all MTC are hereditary, and generally they are identified by a careful family history. However, some familial MTCs are not easily detected by this means and underdiagnosis of MEN 2A is suspected.
Methods
DNA samples from MEN 2A patients were amplified by PCR. The products were incubated with the restriction enzyme Bst ApI or Bgl I.
The samples were loaded in non-denaturing 10% Polyacrilamyde Gel and run at 120 volts for 40 min. The gels were stained with 10 μg/ml ethidium bromide, and the bands were visualized under a UV lamp.
Results
We developed a PCR-mutagenic method to check the integrity of the three bases of the cysteine 634 codon.
Conclusion
The method can be used to detect inherited mutations in MTC patients without a clear family history. The method is relatively simple to use as a routine test in these patients to decrease the underdiagnosis of MEN 2A. In addition, the assay can be used to screen affected families with any mutation in cysteine 634.
doi:10.1186/1471-2350-3-4
PMCID: PMC115869  PMID: 12033991
11.  PTPRC (CD45) is not associated with multiple sclerosis in a large cohort of German patients 
Background
Since contradictory results have been reported, we reanalysed the 77C→G transition in exon 4 of the protein-tyrosine phosphatase receptor-type C (PTPRC also known as CD45) in a large cohort of German MS patients and controls. Different isoforms of the protein are expressed, depending on alternative splicing of exons 4 (CD45RA), 5 (CD45RB) and 6 (CD45RC) (CD45RO, exons 4–6 spliced out). The 77C→G transition does not change the amino acid sequence, but it is probably part of a motif necessary for splicing leading to the isoform CD45RA. The expression of CD45RA is increased in 77C/G heterozygous individuals. The aim of the study was to clarify the importance of the PTPRC 77C→G transition in our German cohort of MS patients.
Methods
PCR products of exon 4 were digested using endonuclease MspI. The resulting restriction fragments of the wildtype C allele are 198 and 62 bp in length. In the G allele an additional restriction site is present yielding fragments of 114 and 84 bp.
Results
The G allele was identified in 10 of the 347 controls (1.4%) and in 7 of 454 MS patients (0.8%; Table 1). No homozygous individuals were found either in the control or in the patient group. Genetic association between the PTPRC 77C→G transition and MS susceptibility was excluded in the MS cohort. In addition, subgrouping patients according to differences in the clinical course of MS or according to HLA-DRB1*15 status did not yield significant differences.
Conclusions
The 77C→G transition in exon 4 of the PTPRC gene may contribute to MS susceptibility only in very few families, if at all, but it is not relevant for the majority of MS cases, including virtually all German patients.
doi:10.1186/1471-2350-3-3
PMCID: PMC116441  PMID: 12028593
12.  A role for the collagen I/III and MMP-1/-13 genes in primary inguinal hernia? 
Background
Abnormal collagen metabolism is thought to play an important role in the development of primary inguinal hernia. This is underlined by detection of altered collagen metabolism and structural changes of the tissue in patients with primary inguinal hernia. However, it is still unknown whether these alterations reflect a basic dysfunction of the collagen synthesis, or of collagen degradation.
Methods
In the present study, we analysed type I and type III procollagen messenger ribonucleic acid (mRNA) and MMP-1 and MMP-13 mRNA in cultured fibroblasts from the skin of patients with primary inguinal hernia, and from patients without hernia (controls) by reverse transcription polymerase chain reaction (RT-PCR) and Northern Blot.
Results
The results indicated that the ratio of type I to type III procollagen mRNA was decreased in patients with primary hernia, showing significant differences as compared to controls (p = 0.01). This decrease was mainly due to the increase of type III procollagen mRNA. Furthermore, RT-PCR analysis revealed that the expression of MMP-1 mRNA in patients with primary hernia is equivalent to that of controls (p > 0.05). In addition, MMP-13 mRNA is expressed neither in patients with primary hernia nor in controls.
Conclusion
We concluded that abnormal change of type I and type III collagen mRNAs contribute to the development of primary inguinal hernia, whereas the expressions of MMP-1 and MMP-13 mRNA appears not to be involved in the development of primary inguinal hernia. Thus, the knowledge on the transcriptional regulation of collagen in patients with primary inguinal hernia may help to understand the pathogenesis of primary inguinal hernia, and implies new therapeutic strategies for this disease.
doi:10.1186/1471-2350-3-2
PMCID: PMC65699  PMID: 11872152
13.  The Siblings With Ischemic Stroke Study (SWISS) Protocol 
Background
Family history and twins studies suggest an inherited component to ischemic stroke risk. Candidate gene association studies have been performed but have limited capacity to identify novel risk factor genes. The Siblings With Ischemic Stroke Study (SWISS) aims to conduct a genome-wide scan in sibling pairs concordant or discordant for ischemic stroke to identify novel genetic risk factors through linkage analysis.
Methods
Screening at multiple clinical centers identifies patients (probands) with radiographically confirmed ischemic stroke and a family history of at least 1 living full sibling with stroke. After giving informed consent, without violating privacy among other family members, the proband invites siblings concordant and discordant for stroke to participate. Siblings then contact the study coordinating center. The diagnosis of ischemic stroke in potentially concordant siblings is confirmed by systematic centralized review of medical records. The stroke-free status of potentially discordant siblings is confirmed by validated structured telephone interview. Blood samples for DNA analysis are taken from concordant sibling pairs and, if applicable, from 1 discordant sibling. Epstein-Barr virus-transformed lymphoblastoid cell lines are created, and a scan of the human genome is planned.
Discussion
Conducting adequately powered genomics studies of stroke in humans is challenging because of the heterogeneity of the stroke phenotype and the difficulty of obtaining DNA samples from clinically well-characterized members of a cohort of stroke pedigrees. The multicentered design of this study is intended to efficiently assemble a cohort of ischemic stroke pedigrees without invoking community consent or using cold-calling of pedigree members.
doi:10.1186/1471-2350-3-1
PMCID: PMC79001  PMID: 11882254

Results 1-13 (13)