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F Giannelli

Publications and source records attributed to F Giannelli.

At least 55 records · Page 3Linked to original sources
PubMed1994

Mutation detection by fluorescent chemical cleavage: application to hemophilia B.

Chemical mismatch detection when combined with PCR or RT-PCR amplification represents one of the most efficient mutation screening methods. This procedure has been used for the analysis of both large populations of mutants and large and complex genes because it detects and locates virtually all sequence variations in segments of DNA of up to 1.7 kb cleaving at, and/or adjacent to, mismatches in heteroduplexes formed by target and probe DNA. By using fluorescent tags and an automatic gel scanning system, the efficiency of the method has been increased fourfold with gains also in the safety and quality control of probes and in the flexibility of the procedure. Using 12 hemophilia B patients, 8 with known mutations and 4 chosen at random, we show how the chemical mismatch cleavage of up to four DNA segments can be multiplexed and examined in a single gel lane and how the increase in efficiency thus obtained can be used either totally to maximize the DNA screened per lane or partly to locate the mutation unequivocally so that only one sequencing reaction is needed to characterize it fully.

DNA Mutational Analysis↗
PubMed1994

First report on UK database of haemophilia B mutations and pedigrees. UK Haemophilia Centres.

In order to obtain complete success in the carrier and prenatal diagnoses required for genetic counselling in haemophilia B a new strategy is being implemented in the UK. This entails the construction of a national confidential database of mutations, pedigrees and haematological data. This will allow the inefficient indirect tests based on the analysis of DNA polymorphisms to be abandoned and direct detection of the gene defect to be used instead. After two and a half years of nationwide collaboration, 702 samples have been collected from 313 families, representing more than half of the UK haemophilia B families, and 217 mutations have been characterised. The 141 diagnostic tests so far performed have clearly indicated that the new strategy not only allows virtually 100% diagnostic success, but also rapid results. This work on haemophilia B may represent a model for other diseases with high mutational heterogeneity.

Databases, Factual↗
PubMed1993

Haemophilia B caused by a missense mutation in the prepeptide sequence of factor IX.

In the course of analysing mutation in the factor IX gene from 200 haemophilia B patients in Sweden and the UK, we have identified one patient with a prepeptide missense mutation. He has severe, antigen negative haemophilia, and complete analysis of his coding sequence reveals a single base transversion (A-->T) causing substitution of isoleucine by asparagine at position -30. This change disrupts the hydrophobic core of the prepeptide, a feature which is required for secretion. Thus, haemophilia in this patient is caused by a failure to secrete factor IX from the hepatocytes.

Amino Acid Sequence↗
PubMed1993

Vertical integration of cosmid and YAC resources for interval mapping on the X-chromosome.

The vertical integration of cosmid and yeast artificial chromosome (YAC) resources is of particular importance in the development of high-resolution maps of selected regions of the human genome. A resource of approximately 95,000 cosmids constructed using DNA from primary fibroblasts of karyotype 49,XXXXX was validated by detailed characterization of a 200-kb cosmid contig spanning exons 8-20 of the dystrophin gene. This resource was used to construct contigs in 0.65 Mb of Xq26 by hybridization of gel-purified YAC DNA to high-density gridded arrays of the cosmid library; positive cosmids were overlapped by finger-printing. Contigs were oriented and ordered relative to existing YACs in the region using cross-hybridization. The overlaps between a representative set of cosmids define 54 intervals of 5-20 kb and were used to construct a high-resolution cosmid interval map of the region, locating markers, dinucleotide repeats, and candidate CpG islands. This approach can be applied rapidly to large regions of the genome and without recourse to subcloning of individual YACs.

Aneuploidy↗
PubMed1993

Analysis of factor VIII mRNA reveals defects in everyone of 28 haemophilia A patients.

Haemophilia A is a mutationally heterogeneous disease caused by defects in the large and complex factor VIII gene. Recent studies examining the putative promoter, all exons and most intron/exon boundaries have failed to detect mutations in half the patients with severe disease leading to hypotheses such as mutations in remote controlling regions or even in genes other than factor VIII. We have amplified the factor VIII gene (putative promotor, coding region and polyadenylation/cleavage signal region) in 8 fragments from reverse transcribed mRNA and genomic DNA. Any mutation is then located by chemical mismatch detection and characterised by direct sequencing. This rapid and efficient method has been fully successful and has revealed an unusual cluster of mutations causing severe disease. Of the 28 patients we have reported, 5 had mild or moderate disease and all had a missense mutation. Twenty-three patients were severely affected and 13 of these had different detrimental mutations that were fully characterised at the genomic DNA level. The remaining 10 patients all had mRNA with exon 22 not contiguous to exon 23. Since all exons were normal and so were the splice sites of intron 22, the mutation in these patients should be in the regions of intron 22 that were not screened. These results prove that all haemophilia A cases are due to mutations of the factor VIII gene where, unexpectedly, intron 22 seems to be the target of approximately 40% of the mutations causing severe haemophilia A.

Amino Acid Sequence↗
PubMed1993

Determination of the organisation of coding sequences within the iduronate sulphate sulphatase (IDS) gene.

The structure of the gene coding for iduronate sulphate sulphatase (IDS) has been determined. We have used exon to exon and vectorette PCR to identify 9 exons within the IDS gene and to characterise the surrounding intron sequences. The results of this study will be useful for the complete analysis of many mutations giving rise to Hunter syndrome. IDS is the first member of the group of lysosomal nonarylsulphatase genes for which the gene structure has been determined. It bears no relationship to the exon organisation of steroid sulphatase, despite the homology between these two proteins. This suggests that the division of the sulphatases into the two subgroups on the basis of substrate specificity is also reflected at the level of gene structure.

Base Sequence↗
PubMed1993

Characteristic mRNA abnormality found in half the patients with severe haemophilia A is due to large DNA inversions.

Surprisingly half of all severe haemophilia A patients have no mutation in the promoter, coding sequences and normal RNA processing signals of the factor VIII gene. Instead they manifest a unique mRNA defect that prevents the amplification of the message across the boundary between exon 22 and 23. This locates the defect to internal regions of intron 22. Novel sequences 3' to exon 22 were isolated from the 9 available patients with the above abnormality by combining RACE and vectorette amplifications on trace amounts of mRNA. This showed that exons 1-22 of the factor VIII mRNA had become part of a hybrid message containing new multi exonic sequences expressed in normal cells. The novel sequences were not located in a YAC covering the whole factor VIII gene. Southern blots from patients probed by novel sequences and clones covering intron 22 showed no obvious abnormalities. This suggested inversions involving intron 22 repeated sequences. Screening of 3 YAC libraries with the novel sequences located them at least 200 kb telomeric (5') to factor VIII and pulsed field gel analysis detected abnormal bands in patients. This demonstrates that the mutations in the patients are inversions of long DNA regions possibly involving the repeated sequences and occurring at the surprising rate of approximately 4 x 10(-6) per gene per gamete per generation.

Base Sequence↗
PubMed1992

Factor VIII gene explains all cases of haemophilia A.

Using an mRNA-based method to examine haemophilia A mutations we provide an explanation for the puzzling report that half of the mutations causing severe disease are not detected by analysis of the putative promoter, exons, and most exon/intron boundaries of the factor VIII gene. An unusual cluster of mutations involving regions of intron 22 not examined earlier leads to defective joining of exons 22 and 23 in the mRNA and caused haemophilia A in 10/24 severely affected UK patients.

Electrophoresis, Agar Gel↗
PubMed1992

Haplotype analysis of identical factor IX mutants using PCR.

We have detected the mutations in the factor IX genes from all of the haemophilia B patients registered at Malmö haemophilia centre and are currently examining the entire UK haemophilia B population. From these studies we have found 13 base substitutions which have recurred in 1-6 other, presumably unrelated, patients. In order to determine the minimum number of independent repeats of each mutation we have used PCR to examine the five factor IX polymorphisms forming the most informative combinations and we have characterised the haplotype of each patient. Patients with different haplotypes are assumed to be unrelated and thus to carry independent mutations. All but one of the 13 mutations occur in at least 2 haplotypes thus pinpointing 12 mutational hotspots and mutations that can be clearly considered detrimental. Two of the 13 substitutions occur at non-CpG sites.

Amino Acid Sequence↗
PubMed1992

Direct estimate of the haemophilia B (factor IX deficiency) mutation rate and of the ratio of the sex-specific mutation rates in Sweden.

Mutation rates for X-linked recessive diseases have so far been estimated indirectly by postulating an equilibrium between the loss of defective genes caused by the low reproductive fitness of affected males and the gain resulting from new mutations. Here, for the first time, we directly estimate both the overall and sex-specific mutation rates for haemophilia B by detecting the gene defect of the families registered at the Malmö Haemophilia Centre. These represent a complete sample of the Swedish haemophilia B population (45 out of 77 pedigrees) and contain 23 families with a single affected male. Fifteen of these males had mothers available for study, and of these mothers, 13 had parents available for study. We show that 3 of the above patients and 10 of their mothers carry new mutations, and by extrapolation calculate that 8 males and 98 females should carry new haemophilia B mutations in the Swedish population (8.52 x 10(6) individuals). This leads to the following estimate of the mutation rates: overall mu = 4.1 x 10(-6); male specific nu = 2.1 x 10(-5); and female specific mu = 1.9 x 10(-6). The ratio of such male to female specific mutation rates is thus nu/mu = 11.

Factor IX↗
PubMed1992

Detection of point mutations and a gross deletion in six Hunter syndrome patients.

We have used screening with the polymerase chain reaction and chemical mismatch detection of amplified cDNA to detect and characterize deletions and point mutations in six Hunter Syndrome patients. A high degree of mutational heterogeneity was observed. The first patient is completely deleted for the gene coding for alpha-L-iduronate sulfate sulfatase, while the second has a point mutation that creates a stop codon. The third patient shows a point mutation that creates a novel splice site that is preferentially utilized and results in partial loss of one exon in the RNA. Patients 4, 5, and 6 have point mutations resulting in single amino acid substitutions. Four of the six single-base changes observed in this study were examples of transitions of the highly mutable dinucleotide CpG to TpG. This study has demonstrated a procedure capable of detecting all types of mutation that affect the function of the IDS protein and should enable direct carrier and prenatal diagnosis for Hunter syndrome families.

Amino Acid Sequence↗
PubMed1992

Construction of a 2.6-Mb contig in yeast artificial chromosomes spanning the human dystrophin gene using an STS-based approach.

A sequence tagged site (STS)-based approach has been used to construct a 2.6-Mb contig in yeast artificial chromosomes (YACs) spanning the human dystrophin gene. Twenty-seven STSs were used to identify and overlap 34 YAC clones. A DNA fingerprint of each clone produced by direct Alu-PCR amplification of YAC colonies and the isolation of YAC insert ends by vectorette PCR were used to detect overlaps in intron 1 (280 kb) where no DNA sequence data were available, thereby achieving closure of the map. This study has evaluated methods for mapping large regions of the X chromosome and provides a valuable resource of the dystrophin gene in cloned form for detailed analysis of gene structure and function in the future.

Base Sequence↗
PubMed1992

The development and application of automated gridding for efficient screening of yeast and bacterial ordered libraries.

An automated gridding procedure for the inoculation of yeast and bacterial clones in high-density arrays has been developed. A 96-pin inoculating tool compatible with the standard microtiter plate format and an eight-position tablet have been designed to fit the Biomek 1000 programmable robotic workstation (Beckman Instruments). The system is used to inoculate six copies of 80 x 120-mm filters representing a total of approximately 20,000 individual clones in approximately 3 h. High-density arrays of yeast artificial chromosome (YAC) and cosmid clones have been used for rapid large-scale hybridization screens of ordered libraries. In addition, an improved PCR library screening strategy has been developed using strips cut from the high-density arrays to prepare row and column DNA pools for PCR analysis. This strategy eliminates the final hybridization step and allows identification of a single clone by PCR in 2 days. The development of automated gridding technology will have a significant impact on the establishment of fully versatile screening of ordered library resources for genomic studies.

Animals↗
PubMed1992

Antenatal diagnosis of haemophilia B by amplification and electrophoresis of an exon fragment with a short deletion.

Carrier identification and antenatal diagnosis were performed in 2 sisters by electrophoretic separation of the normal and abnormal bands obtained after amplification of a fragment of exon h in the factor IX gene. The mutation in the family had been characterised as an 8-base pair (bp) deletion in exon h. By amplification of a 326 bp fragment containing the site of deletion, the shorter 318 bp band of the haemophilia B gene could be separated by electrophoresis of the fragments. The comprehensive data collection at the Haemophilia Centre is of vital importance in the genetic counselling of haemophilia families, and was a crucial step for the successful diagnoses in these sisters.

Adult↗
PubMed1992

Origin of mutation in sporadic cases of haemophilia-B.

Of the 45 haemophilia-B patients registered at the haemophilia centre in Malmö, Sweden, 24 are the sole members of their families to be affected, and in 13 of these 24 cases, ascendant relatives are available for study. Detection of the gene defect showed the mutation to be de novo in the proband in 3 of these 13 cases, and inherited from a carrier mother in the remaining 10 cases. All 10 carrier mothers were shown to have de novo mutations, as the patients' grandfathers were phenotypically and/or haematologically normal, and the grandmothers were non-carriers. Seven restriction fragment length polymorphisms (RFLPs) of the factor IX gene were used to determine whether the de novo mutations of the 10 carrier mothers were of paternal or maternal origin. In 6/10 cases, the RFLP patterns were informative, and indicated the mutation to be of paternal origin.

Adolescent↗