Novel germline hMSH2 genomic deletion and somatic hMSH2 mutations in a hereditary nonpolyposis colorectal cancer family

Germline and somatic mutations of the hMSH2 gene were determined in a Japanese hereditary nonpolyposis colorectal cancer (HNPCC) family fulfilling the Amsterdam criteria. PCR-SSCP-sequencing of genomic DNA detected a somatic hMSH2 mutation of an A deletion at codon 227-229 in a duodenal carcinoma and a somatic hMSH2 mutation of an A insertion at codon 21 in a gastric carcinoma from affected family members, both carcinomas exhibiting high microsatellite instability. However, no germline hMSH2 mutation was detected by the PCR-SSCP-sequencing method. Genomic DNA was then analyzed by Southern blot hybridization using three hMSH2 cDNA probes (probe A involving exons 1-5, probe B involving exons 4-11 and probe C involving exons 9-16) after digestion by restriction enzymes, EcoRI, HindIII and NsiI. The NsiI digest of DNA from normal tissues of affected members exhibited an aberrant 8.6 kb restriction fragment, in addition to the normal 10.6 kb fragment, when hybridized to probes A and B. This suggested the presence of a heterozygous 2kb genomic deletion encompassing exon 4, 5 or 6. RT-PCR-sequencing revealed that the deleted region encompassed exon 5. This novel genomic deletion of the hMSH2 gene was confirmed to be pathogenic, and the Southern hybridization pattern was applied to the pre-symptomatic diagnosis.     

An insertion within the factor IX gene: hemophilia BEl Salvador.

A patient with moderate to severe hemophilia B has been found to have a large insertion within his factor IX gene. The site of insertion is located in a DNA segment of approximately 0.8 kb between exon IV and an EcoRI site within intron D. The size of the DNA insertion is approximately 6 kb, and it contains at least two TaqI sites, two EcoRI sites, and one HindIII site. The insert probably originates from outside the FIX gene and does not represent an internal duplication. We propose that this abnormal FIX gene be called FIX El Salvador in recognition of the birthplace of the patient.     

Molecular analysis of hemophilia A mutations in the Finnish population

We have examined the Finnish hemophilia A population for factor VIII gene mutations. This study included 83 unrelated patients and revealed 10 mutations associated with hemophilia. Using cloned cDNA, genomic, and oligonucleotide probes, we have identified three classes of mutations: five mutations causing the loss of TaqI restriction sites, a point mutation resulting in a new TaqI site, and four partial gene deletions. Although exons 5 and 6 were involved in three of the four partial gene deletions, the extent of the DNA lost differs in each case. The fourth deletion was located entirely within intron 1 and segregated with the disease in a large hemophilia pedigree. There was no history of hemophilia in eight of the 10 families. The origin of the mutation was determined in six of these pedigrees, two of which showed evidence for maternal mosaicism.     

Factor IXMadrid 2: a deletion/insertion in factor IX gene which abolishes the sequence of the donor junction at the exon IV-intron d splice site.

DNA from a patient with severe hemophilia B was evaluated by RFLP analysis, producing results which suggested the existence of a partial deletion within the factor IX gene. The deletion was further localized and characterized by PCR amplification and sequencing. The altered allele has a 4,442-bp deletion which removes both the donor splice site located at the 5' end of intron d and the two last coding nucleotides located at the 3' end of exon IV in the normal factor IX gene; this fragment has been replaced by a 47-bp sequence from the normal factor IX gene, although this fragment has been inserted in inverted orientation. Two homologous sequences have been discovered at the ends of the deleted DNA fragment.     

Structure and restriction fragment length polymorphism of genes for human liver arylamine N-acetyltransferases.

Genomic DNA clones coding for polymorphic and monomorphic arylamine N-acetyltransferases (NAT) of human liver were isolated from a genomic DNA library, and their restriction maps and partial nucleotide sequences were determined. Messenger RNA for monomorphic NAT was coded in one exon, while mRNA for polymorphic NAT was coded in two exons; the 5'-noncoding region was located in one exon 8 kb upstream from another exon containing the coding and 3'-noncoding regions. Recently, we have shown that there are three types of polymorphic NAT gene; one of the genes corresponds to a high NAT activity, while the other two genes give rise to a low NAT activity. The restriction fragment length polymorphism (RFLP) was analyzed by Southern blot hybridization of genomic DNAs from homozygotes of the three polymorphic NAT genes using various fragments of the cloned NAT gene. RFLPs of polymorphic NAT gene were observed in coding and 3'-flanking region upon digestion with BamHI and KpnI.     

The rates and patterns of deletions in the human factor IX gene.

Deletions are commonly observed in genes with either segments of highly homologous sequences or excessive gene length. However, in the factor IX gene and in most genes, deletions (of > or = 21 bp) are uncommon. We have analyzed DNA from 290 families with hemophilia B (203 independent mutations) and have found 12 deletions > 20 bp. Eleven of these are > 2 kb (range > 3-163 kb), and one is 1.1 kb. The junctions of the four deletions that are completely contained within the factor IX gene have been determined. A novel mutation occurred in patient HB128: the data suggest that a 26.8-kb deletion occurred between two segments of alternating purines and pyrimidines and that a 2.3-kb sense strand segment derived from the deleted region was inserted. For our sample of 203 independent mutations, we estimate the "baseline" rates of deletional mutation per base pair per generation as a function of size. The rate for large (> 2 kb) deletions is exceedingly low. For every mutational event in which a given base is at the junction of a large deletion, there are an estimated 58 microdeletions (< 20 bp) and 985 single-base substitutions at that base. Analysis of the nine reported deletion junctions in the factor IX gene literature reveals that (i) five are associated with inversions, orphan sequences, or sense strand insertions; (ii) four are simple deletions that display an excess of short direct repeats at their junctions; (iii) there is no dramatic clustering of junctions within the gene; and (iv) with the exception of alternating purines and pyrimidines, deletion junctions are not preferentially associated with repetitive DNA.     

Recombinations between Alu repeat sequences that result in partial deletions within the C1 inhibitor gene

Genomic DNA sequence analysis was used to define the extent of deletions within the C1 inhibitor gene in two families with type I hereditary angioneurotic edema. Southern blot analysis initially indicated the presence of the partial deletions. One deletion was approximately 2 kb and included exon VII, whereas the other was approximately 8.5 kb and included exons IV–VI. Genomic libraries from an affected member of each family were constructed and clones containing the deletions were analyzed. Sequence analysis of the deletion joints of the mutants and corresponding regions of the normal gene in the two families demonstrated that both deletion joints resulted from recombination of two Alu repetitive DNA elements. Alu repeat sequences from introns VI and VII combined to make a novel Alu in family A, and Alu sequences in introns III and VI were spliced to make a new Alu in family B. The splice sites in the Alu sequences of both mutants were located in the left arm of the Alu element, and both recombination joints overlapped one of the RNA polymerase III promoter sequences. Because the involved Alu sequences, in both instances, were oriented in the same direction, unequal crossingover is the most likely mechanism to account for these mutations.     

Structure of the human laminin B2 chain gene reveals extensive divergence from the laminin B1 chain gene

The exon-intron structure of the human laminin B2 chain gene was elucidated from genomic lambda phage clones spanning 2 kilobase pairs (kb) of the 5'-flanking region, 58 kb of the structural gene and 10 kb of the 3'-flanking region. The entire gene was shown to contain 28 exons. The promoter region has no TATA or CAAT boxes whereas it contains five GC boxes and three AP-2-like binding sites. Comparison with the promoter region of the mouse gene revealed six highly conserved sequences of 14 to 42 base pairs in length. Sequencing of the last exon of the gene showed that the 3'-untranslated region of the mRNA can be up to 2797 nucleotides with five AATAAA potential polyadenylation signals. The similarity of the human 3'-untranslated sequence with that of mouse was shown to be 68.8%. The exon-intron structure of the laminin B2 chain gene demonstrated extensive divergence from the human laminin B1 chain gene, which has 34 exons. Only three intron locations are conserved in these two genes. The overall exon profile of the laminin B2 chain gene correlates only marginally with the pattern of structural domains and internal cysteine-rich repeats in the laminin B2 polypeptide chain.     

Nonhomologous recombination in the human genome: deletions in the human factor VIII gene

Four deletions in the human factor VIII gene have been characterized at the sequence level in patients with hemophilia A. Deletion JH 1 extends 57 kb from IVS 10 to IVS 18. Intron 13 and exon 14 are partially deleted in patients JH 7 and JH 37, with a loss of 3.2 and 2.4 kb of DNA, respectively. The 3' deletion breakpoint of the JH 21 event resides in intron 3 and extends 5' into intron 1, resulting in the loss of exons 2 and 3. Seven of the eight breakpoints sequenced (5' and 3' for each of the four deletions) occur in nonrepetitive sequence, while the 3' breakpoint of the JH 1 resides in an Alu repetitive element. All of the deletions are the result of nonhomologous recombination. The 5' and 3' breakpoints of JH 1, JH 7, and JH 37 share 2- to 3-bp homologies at the deletion junctions. In contrast, two nucleotides have been inserted at the JH 21 deletion junction. Short sequence homologies may facilitate end-joining reactions in nonhomologous recombination events.     

Low frequency of CYP2B deletions in Brazilian patients with congenital adrenal hyperplasia due to 21-hydroxylas deficiency

The frequency of large mutations was determined in 131 Brazilian patients with different clinical forms of 21-hydroxylase deficiency, belonging to 116 families. DNA samples were examined by Southern blotting hybridization with genomic CYP21 and C4cDNA probes after Taql and Bg/II restriction. Large gene conversions were found in 6.6% and CYP21B deletions in 4.4% of the alleles. The breakpoint in these hybrid genes occurred after exon 3 in 92% of the alleles. All rearrangements involving CYP21B gene occurred in the heterozygous form, except in a patient with simple virilizing form who presented homozygous CYP21B deletion. Our data showed that in these Brazilian patients, CYP21B deletions were less frequent than in most of the large series previously reported.     

Complete sequence of the 23-kilobase human COL9A3 gene. Detection of Gly-X-Y triplet deletions that represent neutral variants.

We report the complete sequence of the human COL9A3 gene that encodes the alpha3 chain of heterotrimeric type IX collagen, a member of the fibril-associated collagens with interrupted triple helices family of collagenous proteins. Nucleotide sequencing defined over 23,000 base pairs (bp) of the gene and about 3000 bp of the 5'-flanking sequences. The gene contains 32 exons. The domain and exon organization of the gene is almost identical to a related gene, the human COL9A2 gene. However, exon 2 of the COL9A3 gene codes for one -Gly-X-Y- triplet less than exon 2 of the COL9A2 gene. The difference is compensated by an insertion of 9 bp coding for an additional triplet in exon 4 of the COL9A3 gene. As a result, the number of -Gly-X-Y- repeats in the third collagenous domain remains the same in both genes and ensures the formation of an in-register triple helix. In the course of screening this gene for mutations, heterozygosity for separate 9-bp deletions within the COL1 domain were identified in two kindreds. In both instances, the deletions did not co-segregate with any disease phenotype, suggesting that they were neutral variants. In contrast, similar deletions in triple helical domain of type I collagen are lethal. To study whether alpha3(IX) chains with the deletion will participate in the formation of correctly folded heterotrimeric type IX collagen, we expressed mutant alpha3 chains together with normal alpha1 and alpha2 chains in insect cells. We show here that despite the deletion, mutant alpha3 chains were secreted as heterotrimeric, triple helical molecules consisting of three alpha chains in a 1:1:1 ratio. The results suggest that the next noncollagenous domain (NC2) is capable of correcting the alignment of the alpha chains, and this ensures the formation of an in-register triple helix.     

High resolution deletion breakpoint mapping in the DMD gene by whole cosmid hybridization.

The locus DXS269 (P20) defines a deletion hotspot in the distal part of the Duchenne Muscular Dystrophy gene. We have cloned over 90 kilobase-pairs of genomic DNA from this region in overlapping cosmids. The use of whole cosmids as probes in a competitive DNA hybridization analysis proves a fast and convenient method for identifying rearrangements in this region. A rapid survey of P20-deletion patients is carried out to elucidate the nature of the propensity to deletions in this region. Using this technique, deletion breakpoints are pinpointed to individual restriction fragments in patient DNAs without the need for tedious isolation of single copy sequences. Simultaneously, the deletion data yield a consistent restriction map of the region and permit detection of several RFLPs. A 176 bp exon was identified within the cloned DNA, located 3' of an intron exceeding 150 Kb in length. Its deletion causes a frameshift in the dystrophin reading frame and produces the DMD phenotype. This exon is one of the most frequently deleted exons in BMD/DMD patients and its sequence is applied in a pilot study for diagnostic deletion screening using Polymerase Chain Reaction amplification.     

Nullisomic deletion of the mcf.2 transforming gene in two haemophilia B patients.

The mcf.2 transforming gene sequence has been located to the region between 29 and 61 kb 3' of the factor IX gene. Two unrelated haemophilia B patients who raise antibodies to infused factor IX ('inhibitors') have deletions in excess of 273 kb encompassing the factor IX and mcf.2 genes and a CG-rich island. We believe these patients show the first nullisomic deletion of a transforming gene to be reported. No clinical condition can be attributed to the loss of the mcf.2 gene.     

A complete deletion of the factor IX gene and new TaqI variant in a hemophilia B kindred

Summary We report a hemophilia B kindred in which the proband has a complete deletion of the factor IX gene extending a minimum of 80 kilobase pairs (kb) 3 of the gene. This individual has severe factor IX deficiency with no detectable circulating factor IX protein. In common with one previous report, despite a total deletion of the factor IX gene, this patient has not developed antibodies to factor IX. The mother of the proband was found to have a new TaqI variant of the factor IX gene on the nondeletion-bearing X chromosome. The location of the altered TaqI site was found to be 5 of exon IV between residues 9731-9734 and does not affect the function of the factor IX protein. The familial natures of both the variant allele and the deletion were established. In addition a study of this kindred at the DXS99 locus demonstrated the first reported recombination event between this site and the factor IX gene.     

Isolation and characterization of the human UGT2B15 gene, localized within a cluster of UGT2B genes and pseudogenes on chromosome 4

Glucuronidation is a major pathway of androgen metabolism and is catalyzed by UDP-glucuronosyltransferase (UGT) enzymes. UGT2B15 and UGT2B17 are 95% identical in primary structure, and are expressed in steroid target tissues where they conjugate C19 steroids. Despite the similarities, their regulation of expression are different; however, the promoter region and genomic structure of only the UGT2B17 gene have been characterizedX to date. To isolate the UGT2B15 gene and other novel steroid-conjugating UGT2B genes, eight P-1-derived artificial chromosomes (PAC) clones varying in length from 30 kb to 165 kb were isolated. The entire UGT2B15 gene was isolated and characterized from the PAC clone 21598 of 165 kb. The UGT2B15 and UGT2B17 genes are highly conserved, are both composed of six exons spanning approximately 25 kb, have identical exon sizes and have identical exon-intron boundaries. The homology between the two genes extend into the 5'-flanking region, and contain several conserved putative cis-acting elements including Pbx-1, C/EBP, AP-1, Oct-1 and NF/kappaB. However, transfection studies revealed differences in basal promoter activity between the two genes, which correspond to regions containing non-conserved potential elements. The high degree of homology in the 5'-flanking region between the two genes is lost upstream of -1662 in UGT2B15, and suggests a site of genetic recombination involved in duplication of UGT2B genes. Fluorescence in situ hybridization mapped the UGT2B15 gene to chromosome 4q13.3-21.1. The other PAC clones isolated contain exons from the UGT2B4, UGT2B11 and UGT2B17 genes. Five novel exons, which are highly homologous to the exon 1 of known UGT2B genes, were also identified; however, these exons contain premature stop codons and represent the first recognized pseudogenes of the UGT2B family. The localization of highly homologous UGT2B genes and pseudogenes as a cluster on chromosome 4q13 reveals the complex nature of this gene locus, and other novel homologous UGT2B genes encoding steroid conjugating enzymes are likely to be found in this region of the genome.