Identification of FVIII gene mutations in patients with hemophilia A using new combinatorial sequencing by hybridization.

BACKGROUND: Standard methods of mutation detection are time consuming in Hemophilia A (HA) rendering their application unavailable in some analysis such as prenatal diagnosis. OBJECTIVES: To evaluate the feasibility of combinatorial sequencing-by-hybridization (cSBH) as an alternative and reliable tool for mutation detection in FVIII gene. PATIENTS/METHODS: We have applied a new method of cSBH that uses two different colors for detection of multiple point mutations in the FVIII gene. The 26 exons encompassing the HA gene were analyzed in 7 newly diagnosed Italian patients and in 19 previously characterized individuals with FVIII deficiency. RESULTS: Data show that, when solution-phase TAMRA and QUASAR labeled 5-mer oligonucleotide sets mixed with unlabeled target PCR templates are co-hybridized in the presence of DNA ligase to universal 6-mer oligonucleotide probe-based arrays, a number of mutations can be successfully detected. The technique was reliable also in identifying a mutant FVIII allele in an obligate heterozygote. A novel missense mutation (Leu1843Thr) in exon 16 and three novel neutral polymorphisms are presented with an updated protocol for 2-color cSBH. CONCLUSIONS: cSBH is a reliable tool for mutation detection in FVIII gene and may represent a complementary method for the genetic screening of HA patients.

Glanzmann thrombasthenia secondary to a Gly273-->Asp mutation adjacent to the first calcium-binding domain of platelet glycoprotein IIb.

We studied the defect responsible for Glanzmann thrombasthenia in a patient whose platelets expressed < 5% of the normal amount of GPIIb-IIIa. Genetic and biochemical evidence indicated that the patient's GPIIIa genes were normal. However, DNA analysis revealed the patient homozygous for a G818-->A substitution in her GPIIb genes, resulting in a Gly273-->Asp substitution adjacent to the first GPIIb calcium-binding domain. To determine how this mutation impaired GPIIb-IIIa expression, recombinant GPIIb containing the mutation was coexpressed with GPIIIa in COS-1 cells. The GPIIb mutant formed stable GPIIb-IIIa heterodimers that were not immunoprecipitated by either of two heterodimer-specific monoclonal antibodies, indicating that the mutation disrupted the epitopes for these antibodies. Moreover, the GPIIb in the heterodimers was not cleaved into heavy and light chains, indicating that the heterodimers were not transported from the endoplasmic reticulum to the Golgi complex where GPIIb cleavage occurs, nor were the mutant heterodimers expressed on the cell surface. These studies demonstrate that a Gly273-->Asp mutation in GPIIb does not prevent the assembly of GPIIb-IIIa heterodimers, but alters the conformation of these heterodimers sufficiently to impair their intracellular transport. The impaired GPIIb-IIIa transport is responsible for the thrombasthenia in this patient.

Current perspectives in protein array technology.

This article reviews post-2000 trends in the development of two-dimensional protein microarrays and nanoarrays. Progress in array manufacture, assay design and applications are considered, with an emphasis on issues surrounding the implementation of arrays in clinical diagnostics. These include the effect of factors in the pre-analytical phase (quality of the reagents, sample integrity, etc.), and those in the analytical phase that contribute to inaccuracy and imprecision of an array-based assay. Important requirements for the quality control and quality assurance of protein microarray assays as they move from the research environment into routine clinical application are also discussed.

Parallel molecular genetic analysis.

We describe recent progress in parallel molecular genetic analyses using DNA microarrays, gel-based systems, and capillary electrophoresis and utilization of these approaches in a variety of molecular biology assays. These applications include use of polymorphic markers for mapping of genes and disease-associated loci and carrier detection for genetic diseases. Application of these technologies in molecular diagnostics as well as fluorescent technologies in DNA analysis using immobilized oligonucleotide arrays on silicon or glass microchips are discussed. The array-based assays include sequencing by hybridization, cDNA expression profiling, comparative genome hybridization and genetic linkage analysis. Developments in non microarray-based, parallel analyses of mutations and gene expression profiles are reviewed. The promise of and recent progress in capillary array electrophoresis for parallel DNA sequence analysis and genotyping is summarized. Finally, a framework for decision making in selecting available technology options for specific molecular genetic analyses is presented.

Combined segregation and linkage analysis of inflammatory bowel disease in the IBD1 region using severity to characterise Crohn's disease and ulcerative colitis. On behalf of the GISC.

Inflammatory bowel disease (IBD) is a chronic relapsing disorder affecting the gastro-intestinal tract and is subdivided into two main subtypes: Crohn's disease (CD) and ulcerative colitis (UC). Although the aetiology of IBD is unknown, a strong genetic susceptibility is suggested and different candidate regions have been identified for both CD and UC. The IBD1 region on chromosome 16 has been confirmed to be important for susceptibility to CD, whereas conflicting evidence has been obtained for UC. We performed a combined linkage and segregation analysis in the identified IBD1 region on a sample of 82 extended families with IBD using a parametric method implemented in the computer program COMDS. This approach allows simultaneous evaluation of linkage while estimating the mode of inheritance and to include severity of the trait to characterise the CD and UC phenotypes. Our results are consistent with the presence of a major gene in the IBD1 region close to D16S408 involved in both UC and CD. Furthermore, our data support evidence that a single mutation in the gene leads more frequently to UC, whereas inheritance of two mutant alleles results in the more severe CD. In our study the IBD1 locus was found to have a major role in IBD predisposition in the Italian population.

Simple two-color array-based approach for mutation detection.

The ability to analyze multiple polymorphic/mutation sites rapidly and accurately is pivotal in all areas of genetic analysis. We have applied single nucleotide primer extension (SNE) for detection of multiple point mutations in a micro-array format using two-color, fluorescent dye-tagged dideoxynucleoside triphosphate terminators (ddNTPs). The oligonucleotide primer ending one nucleotide short of the mutation site being probed is bound to the slide and single-base extended in place with two different Cy5/Cy3 dye-tagged terminators using solution-phase, locus-specific, single-stranded complementary templates generated by PCR from genomic DNA. The composite fluorescence produced contains peaks of distinct wave lengths corresponding to each Cy dye-tagged terminator incorporated, resulting in a fluorescent 'fingerprint' for each DNA target. DNA polymerase-catalyzed incorporation of Cy dye-tagged dideoxynucleoside triphosphates was dependent on the particular dyes, the specific ddNTP, the DNA target concentration, sequence of the template, on-slide temperature cycling and washing conditions. Results from analysis of mutations in the human hemochromatosis and connexin 26 genes show that this approach has several advantages over existing methods and is simple, rapid, robust, cost effective and accurate with potential applications in many areas of genetic analysis.

High carrier frequency of the 35delG deafness mutation in European populations. Genetic Analysis Consortium of GJB2 35delG.

Congenital deafness accounts for about 1 in 1000 infants and approximately 80% of cases are inherited as an autosomal recessive trait. Recently, it has been demonstrated that connexin 26 (GJB2) gene is a major gene for congenital sensorineural deafness. A single mutation (named 35delG) was found in most recessive families and sporadic cases of congenital deafness, among Caucasoids, with relative frequencies ranging from 28% to 63%. We present here the analysis of the 35delG mutation in 3270 random controls from 17 European countries. We have detected a carrier frequency for 35delG of 1 in 35 in southern Europe and 1 in 79 in central and northern Europe. In addition, 35delG was detected in five out of 376 Jewish subjects of different origin, but was absent in other non-European populations. The study suggests either a single origin for 35delG somewhere in Europe or in the Middle East, and the possible presence of a carrier advantage together with a founder effect. The 35delG carrier frequency of 1 in 51 in the overall European population clearly indicates that this genetic alteration is a major mutation for autosomal recessive deafness in Caucasoids. This finding should facilitate diagnosis of congenital deafness and allow early treatment of the affected subjects.

Linkage of DFNB1 to non-syndromic neurosensory autosomal-recessive deafness in Mediterranean families.

Recent studies show a susceptibility locus (DFNB1) responsible for non-syndromic neurosensory autosomal-recessive deafness (NSRD) mapping to the pericentromeric region of chromosome 13q. In order to better understand the frequency with which DFNB1 is the gene for deafness in our patient population and the role of DFNB1 in Caucasians, we performed a genetic linkage study with four microsatellite markers linked to DFNB1 in a total of 48 independent Mediterranean families, of which 30 and 18 were of Italian and Spanish descent, respectively. A maximum two-point lod score of 7.28 was found with marker D13S115 at a recombination frequency of theta 0.1. Significant lod scores were also obtained for D13S143, D13S292 and D13S175. Genetic heterogeneity was confirmed using the HOMOG program which indicated absence of linkage to DFNB1 in approximately 21% of the sample. This study clearly demonstrates that DFNB1 plays an important role in 79% of Mediterranean families with NSRD. Furthermore, results from multipoint analysis predict that the DFNB1 gene maps between markers D13S175 and D13S115 which are separated by approximately 14.2 cM.

Genetic analysis in Italian families with inflammatory bowel disease supports linkage to the IBD1 locus--a GISC study.

Epidemiological studies suggest that inherited factors influence susceptibility to inflammatory bowel disease (IBD), and some candidate loci have been described. In order to verify whether the same loci are responsible for predisposition to IBD in our population, we carried out a linkage study in a series of 58 Italian families with Crohn's disease (CD) and ulcerative colitis (UC). HLA-DQ alleles, motilin gene, and 34 microsatellites flanking the previously described loci on chromosomes 3, 6, 7, 12 and 16 were analysed by non-parametric linkage analysis in 16 and 23 families with CD and UC, respectively, and in 19 families where CD and UC coexisted. Non parametric analysis using GENEHUNTER yielded maximum NPL scores for marker D16S408 in all IBD families combined (2.71, P = 0.003), for marker D16S419 in CD (1.97, P = 0.026) and for marker D16S514 in UC families (2.44, P = 0.007). These markers map in the previously described IBD1 region. No significant linkage was found for markers of chromosomes 3, 6, 7 and 12. The present study performed in a Southern European population provides additional support for the conclusion with the IBD1 locus has a clear role in the genetic susceptibility to IBD.

Four-laser scanning confocal system for microarray analysis.

We have constructed a confocal scanner suitable for routine microarray analysis from commercially available parts. We have outlined the details that should be considered when designing such an instrument and listed some of the specific components comprising the system [the full list of system components is available on CD from the corresponding author (D.J.G.) at no charge]. Here, we describe the methods used to test the linearity and sensitivity of the instrument. Performance was evaluated with two commonly used dyes, fluorescein and Cy5. While the instrument had a linear correlation between the dye concentration and fluorescence intensity, the observed deviation from a slope of 1.0 underscores the importance of running multipoint calibration experiments to obtain accurate dye quantitation over the full dynamic range of the scanner. This method has utility in testing commercial instruments in addition to the scanner described here. An array with over 300 spots dyed with Cy3 was scanned with our instrument and a high-end commercial instrument. The agreement between the two instruments was very good over a 1000-fold intensity range. Our scanner is a cost-effective alternative to more costly commercial scanners with similar capabilities.

Duchenne/Becker muscular dystrophy carrier detection using quantitative PCR and fluorescence-based strategies.

Dystrophin gene deletions account for up to 68% of all Duchenne (DMD) and Becker (BMD) muscular dystrophy mutations. In affected males, these deletions can be detected easily using multiplex PCR tests which monitor for exon presence. In addition, quantitative dosage screening can discriminate female carriers. We previously analyzed multiplex PCR products by gel electrophoresis and quantitation of fluorescently labeled primers with the Gene Scanner in order to test carrier status. These multiplex PCR protocols detect DMD gene deletions adequately, but require up to 18 pairs of fluorochrome-labeled primers. We previously described two alternative fluorescent labeling strategies, each with approximately 1,000-fold greater sensitivity than ethidium bromide staining, which can be used to quantify the products of multiplex PCR. The first method uses the DNA intercalating thiazole orange dye TOTO-1 to stain PCR products after 20 cycles. In the second method, fluorescein-12,2'-dUTP is incorporated into products during PCR as a fluorescent tag for subsequent quantitative dosage studies. Both methods label all multiplexed exons including the 506 bp exon 48 fragment that is difficult to detect and quantify by standard ethidium bromide staining. Using this approach, we determined DMD/BMD carrier status in 24 unrelated families using a fluorescent fragment analyzer. Analysis of fluorochrome-labeled PCR products facilitates quantitative multiplex PCR for gene-dosage analysis.

Infundibulopelvic stenosis, multicystic kidney, and calyectasis in a kindred: clinical observations and genetic analysis.

Congenital obstructive anomalies of the urinary tract usually occur sporadically. We describe inheritance in a three-generation kindred of a spectrum of kidney anomalies consistent with an autosomal-dominant mode of transmission, with incomplete penetrance, calyectasis (maternal grandmother), infundibulopelvic stenosis (uncle), and multicystic kidney (male proband, age 4 years). The proband's mother, father and half sister had normal renal imaging studies. Inheritance of informative polymorphic markers (3'-HVR, GGG1, GGG9, SM-7, KG8, and CW3) mapping close to the adult polycystic kidney disease type 1 (PKD-1) and tuberous sclerosis (TSC-2) loci on chromosome 16p was evaluated by Southern blot studies and by PCR-based, fluorescent genotyping for linkage to phenotype. The 3 affected individuals, as well as the unaffected mother (obligate carrier) and unaffected half-sister, inherit a common chromosome haplotype linked to the PKD1 locus. Our findings support the hypothesis that these anomalies may be part of a spectrum of obstructive renal dysplasia which are inherited as a simple Mendelian trait exhibiting an autosomal-dominant mode of transmission with variable expression and incomplete penetrance.

Submicroscopic deletion in cousins with Prader-Willi syndrome causes a grandmatrilineal inheritance pattern: effects of imprinting.

The Prader-Willi syndrome (PWS) critical region on 15q11-q13 is subject to imprinting. PWS becomes apparent when genes on the paternally inherited chromosome are not expressed. Familial PWS is rare. We report on a family in which a male and a female paternal first cousin both have PWS with cytogenetically normal karyotypes. Fluorescence in situ hybridization (FISH) analysis shows a submicroscopic deletion of SNRPN, but not the closely associated loci D15S10, D15S11, D15S63, and GABRB3. The cousins' fathers and two paternal aunts have the same deletion and are clinically normal. The grandmother of the cousins is deceased and not available for study, and their grandfather is not deleted for SNRPN. DNA methylation analysis of D15S63 is consistent with an abnormality of the imprinting center associated with PWS. "Grandmatrilineal" inheritance occurs when a woman with deletion of an imprinted, paternally expressed gene is at risk of having affected grandchildren through her sons. In this case, PWS does not become evident as long as the deletion is passed through the matrilineal line. This represents a unique inheritance pattern due to imprinting.

Rapid sizing of polymorphic microsatellite markers by capillary array electrophoresis.

Genetic mapping and DNA sequencing projects could potentially be completed more rapidly by using capillary array electrophoresis (CAE) systems running 48-96 capillaries simultaneously. Currently, multiplex polymerase chain reaction (PCR) and multicolor fluorescent dye-labeling strategies are used to generate DNA profiles containing 18-24 genotypes per sample. By using 4-color fluorescence detection and these multiplex PCR strategies, a CAE system has the capacity to generate up to 5.5 million genotypes per year. CAE offers extremely fast, high-resolution separation of DNA and more automated sample processing than conventional systems because the labor-intensive slab-gel pouring and sample-loading steps are eliminated. We used a prototype CAE system in an ongoing linkage analysis study of inherited deafness in Mediterranean families. CA-repeat markers linked to deafness susceptibility genes on chromosomes 7, 11 and 13 were analyzed and DNA profiles generated which contain 6 markers per color. Fragment sizes of over 28,000 short tandem repeat alleles and 3200 CA-repeat alleles have been determined by CAE. An average sizing precision of +/- 0.12 base pairs (bp) for fragments up to 350 bp was realized in 1-h runs. In addition, a versatile non-denaturing matrix was used to separate DNA sizing standards, restriction digests, and multiplex PCR samples. Application of this matrix to Duchenne muscular dystrophy exon deletion screening is also described. These CAE approaches should facilitate rapid genotyping of microsatellite markers and subsequent identification of disease-causing mutations.

Microchip-based Devices for Molecular Diagnosis of Genetic Diseases.

Microchips, constructed with a variety of microfabrication technologies (photolithography, micropatterning, microjet printing, light-directed chemical synthesis, laser stereochemical etching, and microcontact printing) are being applied to molecular biology. The new microchip-based analytical devices promise to solve the analytical problems faced by many molecular biologists (eg, contamination, low throughput, and high cost). They may revolutionize molecular biology and its application in clinical medicine, forensic science, and environmental monitoring. A typical biochemical analysis involves three main steps: (1) sample preparation, (2) biochemical reaction, and (3) detection (either separation or hybridization may be involved) accompanied by data acquisition and interpretation. The construction of a miniturized analyzer will therefore necessarily entail the miniaturization and integration of all three of these processes. The literature related to the miniaturization of these three processes indicates that the greatest emphasis so far is on the investigation and development of methods for the detection of nucleic acid, followed by the optimization of a biochemical reaction, such as the polymerase chain reaction. The first step involving sample preparation has received little attention. In this review the state of the art of, microchip-based, miniaturized analytical processes (eg, sample preparation, biochemical reaction, and detection of products) are outlined and the applications of microchip-based devices in the molecular diagnosis of genetic diseases are discussed.

Analysis of 31 CFTR mutations by polymerase chain reaction/oligonucleotide ligation assay in a pilot screening of 4476 newborns for cystic fibrosis.

OBJECTIVES: Molecular biological testing for genetic diseases has grown rapidly, but speed, accuracy, specificity, sensitivity, throughput, and cost become more important as large scale screening is considered. This is a pilot study of an assay for the simultaneous detection of up to 31 cystic fibrosis mutations in a multicentre population based screening of 4476 Italian newborns. METHODS: The assay is a polymerase chain reaction, followed by an oligonucleotide ligation assay (PCR/OLA) and finally a sequence coded separation. It allows the detection of up to 31 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Guthrie spots were used as a source of template DNA. RESULTS: 144 carriers were detected during the analysis of 4476 samples, which translates into a carrier frequency of 1/31.1. Forty two carriers were detected from 1341 samples in Pavia (1/31.9), 53 from 1574 in Turin (1/29.7), and 49 from 1561 in San Giovanni Rotondo (1/31.8). Fifteen different mutations were detected, the most common being delta F508 (0.625). Other common mutations included G542X (16 of 144), which was particularly common in southern Italy (14 of 49), N1303K (8 of 144), and R117H (8 of 144), detected only in the northern centres. CONCLUSIONS: PCR/OLA is a robust, accurate, user friendly method for cystic fibrosis screening of newborns using blood spots in a semiautomated way at a low cost per mutation (0.8 Euro).

A pilot C282Y hemochromatosis screening in Italian newborns by TaqMan technology.

Hereditary hemochromatosis (HH) is a disorder of iron metabolism that leads to iron overload in middle age and can be caused by homozygosity for the C282Y mutation in the HFE gene. Preliminary studies have estimated the frequency of this mutation at 0.5-1% in Italy, but this has not been verified on a large sample. We analyzed 1,331 Italian newborns for the C282Y mutation in the HFE gene using dried blood spots (DBS) from the Neonatal Screening Center in Turin, Italy. The mutation was assessed using a semi-automatable 5'-nuclease assay (TaqMan technology). We detected 55 heterozygotes and no homozygotes in our sampling, resulting in an overall frequency of 2.1% +/- 0.6 for the C282Y allele. Differences in allele frequency were observed, and ranged from 2.7% +/- 1.3 in samples from Northern Italy, to 1.7% +/- 0.9 in samples from Central-Southern Italy. The low frequency of the at-risk genotype for iron overload suggests that genetic screening for HFE in Italy would not be cost effective. The present study, in addition to defining C282Y frequency, documents detection of the major HFE mutation on routine DBS samples from neonatal screening programs using a semi-automatable, rapid, reliable, and relatively inexpensive approach.

Microarray-based genetic analyses for studying susceptibility to arterial and venous thrombotic disorders.

We describe the potential of microarray technology for parallel, high-throughput approaches to molecular detection of DNA variations associated with progression to arterial and venous thrombotic diseases. The use of the newly commercialized NanoChip platform and a framework for genetic screening with a list of potential genetic targets useful to the evaluation of cardiovascular risk are presented. Implementation in clinical setting of analytical silicon and glass microarrays will facilitate early diagnosis, help direct specific advice to high-risk individuals and evaluate treatment strategies.

[Bone marrow aplasia]. / Le aplasie midollari.

Aplastic anaemia is a haematological syndrome in which pancytopenia is due to a depletion, damage of inhibition of hemopoietic stem cells. The pathogenetic factors are still unclear: damage or inhibition of hemopoietic stem cells may be direct or indirect mediated through changes in the cellular humoral environment; evidence is accumulating that in some cases these processes are of autoimmune nature. The prognostic evaluation is based on hematological parameters at diagnosis: severe aplastic anemia (SAA) is defined by the following criteria: neutrophils less than 0.5 X 10(9)/l; reticulocytes less than 0.5 X 10(9)/l and platelets less than 20 X 10(9)/l. Survival rates in children with SAA are poor; the probability of survival at 1 year from diagnosis being 10%. In this form treatment is based on supportive therapy (transfusion and prevention of infections) and on a specific therapy: immunosuppression and/or BMT. BMT is reserved to patient with an HLA identical sibling donor and may be curative in 70-80% of cases. To date the use of antilymphocytoglobulin in SAA has also given satisfactory results with a favorable response in 50-60% of cases.

The retinoblastoma tumor suppressor pathway modulates the invasiveness of ErbB2-positive breast cancer.

The processes that control the progression of ductal carcinoma in situ (DCIS) to invasive breast cancer remain poorly understood. Epidermal growth factor receptor 2 (ErbB2) overexpression is common in DCIS, as is disruption of the retinoblastoma tumor suppressor (RB) pathway. Here, we examined the cooperative impact of ErbB2 and RB deregulation on facets of disease progression. Our studies demonstrate that RB deficiency altered the expression of key molecules needed for proper cellular organization and epithelial cell-cell adhesion as part of a program related to the epithelial-to-mesenchymal transition (EMT). An increase in the invasive potential of ErbB2-overexpressing cells was observed upon RB depletion. Further, stable knockdown of RB resulted in invasive lesions in orthotopic xenograft assays, compared with DCIS-like lesions developing from RB-proficient cells. Conversely, the invasive phenotype observed in ErbB2-positive cancer models was inhibited through CDK4/6 inhibition in an RB-dependent manner. Finally, in a cohort of DCIS cases, we show that, although elevated levels of ErbB2 are associated with increased risk of a subsequent DCIS recurrence, it is not associated with progression to invasive disease. In contrast, RB loss in ErbB2-positive DCIS cases was associated with increased risk for invasive breast cancer. Taken together, these data demonstrate a key role for the RB pathway in invasion associated with breast tumor progression, and shed light on the key molecular events that promote the progression of DCIS to invasive disease.