HLA-A*68020103 shows an eight nucleotides deletion within intron 2 but has normal mRNA splicing and serological recognition

A novel A*68020103 allele was completely characterized by sequencing in a Spanish bone marrow donor. A*68020103 has an eight nucleotides deletion at the 5'-end of intron 2, when compared with other A*6802 alleles. This alteration does not affect either its mRNA splicing process or serological detection.     

Germinal HPRT splice donor site mutation results in multiple RNA splicing products in T-lymphocyte cultures

We have used peripheral blood T-lymphocyte cultures to analyze the hprt mutation in two Lesch-Nyhan syndrome males who are cousins and to confirm the carrier status of female members of the family. Both cDNA and genomic DNA sequencing studies show that this patient carries a hitherto undescribed single base deletion in the exon 5 donor splice site sequence (15:+1, δG, base number 31635). The largest cDNA product contained all nine hprt exons plus an insertion of 66 bases of intron 5, consistent with the use of a cryptic splice site in intron 5 (aag₆₇/gtaagc). This splicing error would result in a chain terminating codon immediately after exon 5 (15:2–4, taa) and predicts a polypeptide of 133 amino acids. This loss of the normal splice donor site also results in multiple hprt mRNA species, combining the use of the cryptic splice site in intron 5 and splicing errors involving exons 2–6. In addition to defining a new Lesch-Nyhan mutation (hprt_Henryville), these results provide insight into aberrant splicing of hprt mRNA in T-lymphocytes.     

Complete sequence analysis of the A*1103 allele

Here we report the full-length sequence of a novel A*11 variant. The variant was identified by ARMS-PCR and serology, the sequence was confirmed by cloning and subsequent sequencing. This variant, A*1103, found in a family of oriental origin, resembles the A*1101 sequence in exon 2 but differs in exon 3 with regard to codons 151 and 152. The polymorphism's result in two amino acid substitutions (one conserved (His->Arg), one introducing a negative charge (Ala->Glu)) located in the alpha2 helical region. The arginine at amino acid position 151 is rare amongst Alocus alleles and is besides A*1103 only observed in A*29 variants, the glutamine at amino acid position 152 is shared with A*0301, A*25, *26, *34 variants and the A*02 subtypes A*0203, *0213 and *0226. In fact, the amino acid motif comprising codons 151 and 152 is unique to A*1103 among Alocus alleles, but is common to C-locus alleles.     

Utilisation of a cryptic non-canonical donor splice site of the gene encoding PARAFIBROMIN is associated with familial isolated primary hyperparathyroidism

More than 99% of all splice sites conform to consensus sequences that usually include the invariant dinucleotides gt and ag at the 5' and 3' ends of the introns, respectively. We report on the utilisation of a non-consensus (non-canonical) donor splice site within exon 1 of the HRPT2 gene in familial isolated primary hyperparathyroidism (FIHP). HRPT2 mutations are more frequently associated with the hyperparathyroidism-jaw tumour syndrome (HPT-JT). Patients with FIHP were identified to have a donor splice site mutation, IVS1+1 g-->a, and the consequences of this for RNA processing were investigated. The mutant mRNA lacked 30 bp and DNA sequence analysis revealed this to result from utilisation of an alternative cryptic non-canonical donor splice site (gaatgt) in exon 1 together with the normally occurring acceptor splice site in intron 1. Translation of this mutant mRNA predicted the in-frame loss of 10 amino acids in the encoded protein, termed PARAFIBROMIN. Thus, these FIHP patients are utilising a ga-ag splice site pair, which until recently was considered to be incompatible with splicing but is now known to occur as a rare (<0.02%) normal splicing variant.     

Mutation screening of the fibrillin-1 (FBN1) gene in 76 unrelated patients with Marfan syndrome or Marfanoid features leads to the identification of 11 novel and three previously reported mutations

Mutations in the gene encoding fibrillin-1 (FBN1) cause Marfan syndrome (MFS) and other related connective tissue disorders. In this study we performed SSCP to analyze all 65 exons of the FBN1 gene in 76 patients presenting with classical MFS or related phenotypes. We report 7 missense mutations, 3 splice site alterations, one indel mutation, one nonsense mutation and two mutations causing frameshifts: a 16bp deletion and a single nucleotide insertion. 5 of the missense mutations (Y1101C, C1806Y, T1908I, G1919D, C2251R) occur in calcium-binding Epidermal Growth Factor-like (EGFcb) domains of exons 26, 43, 46 and 55, respectively. One missense mutation (V449I) substitutes a valine residue in the non-calcium-binding epidermal growth factor like domain (EGFncb) of exon 11. One missense mutation (G880S) affects the "hybrid" motif in exon 21 by replacing glycine to serine. The 3 splice site mutations detected are: IVS1-1G>A in intron 1, IVS38-1G>A in intron 38 and IVS46+5G>A in intron 46. C628delinsK was identified in exon 15 leading to the substitution of a conserved cysteine residue. Furthermore two frameshift mutations were found in exon 15 (1904-1919del ) and exon 63 (8025insC) leading to premature termination codons (PTCs) in exon 17 and 64 respectively. Finally we identified a nonsense mutation (R429X) located in the proline rich domain in exon 10 of the FBN1 gene. Y1101C, IVS46+5G>A and R429X have been reported before.     

A mutation in a rare type of intron in a sodium-channel gene results in aberrant splicing and causes myotonia

Many mutations in the skeletal-muscle sodium-channel gene SCN4A have been associated with myotonia and/or periodic paralysis, but so far all of these mutations are located in exons. We found a patient with myotonia caused by a deletion/insertion located in intron 21 of SCN4A, which is an AT-AC type II intron. This is a rare class of introns that, despite having AT-AC boundaries, are spliced by the major or U2-type spliceosome. The patient's skeletal muscle expressed aberrantly spliced SCN4A mRNA isoforms generated by activation of cryptic splice sites. In addition, genetic suppression experiments using an SCN4A minigene showed that the mutant 5' splice site has impaired binding to the U1 and U6 snRNPs, which are the cognate factors for recognition of U2-type 5' splice sites. One of the aberrantly spliced isoforms encodes a channel with a 35-amino acid insertion in the cytoplasmic loop between domains III and IV of Nav1.4. The mutant channel exhibited a marked disruption of fast inactivation, and a simulation in silico showed that the channel defect is consistent with the patient's myotonic symptoms. This is the first report of a disease-associated mutation in an AT-AC type II intron, and also the first intronic mutation in a voltage-gated ion channel gene showing a gain-of-function defect.     

An HLA-DR11/DQ3 haplotype with a DRB1*0301 sequence motif in the third hypervariable region of the HLA-DR beta-1 chain: molecular and serological analysis of its generation in a European Caucasian family

The formation of a new human leukocyte antigen (HLA)-DRB1 allele (DRB1*0340) has been detected during the routine testing of a European Caucasian blood and potential stem cell donor and his family. HLA typing of the donor with two polymerase chain reaction - sequence specific oligonucleotides (PCR-SSO) systems yielded inconclusive results. HLA typing of the family members including sequence-based typing of DRB1 in both directions after haplotype-specific amplification showed that the allele had most likely formed by a double crossover event in exon 2 of the DRB1 gene. The HLA haplotype containing the new allele was most probably derived from the father, who was typed as HLA-DRB1*0301,*1101 and DRB3*0101,*0202. The comparison of the sequences of the paternal DRB1 and DRB3 alleles with the exon 2 sequence of the DRB1*0340 showed that it had most likely formed through an uptake of at least the sequence part codons 58–77 of DRB1*0301 (donor) by DRB1*1101 (acceptor). We suppose that the recombination sites are located in the sequences from codons 38–57 and codons 78–88. At the protein level, more than 50% of the alpha-helical structure of the DRB1*1101 chain is replaced by a DRB1*0301-derived sequence with the exchange of several amino acids. Serological typing of the allele showed HLA-DR3. However, one monoclonal anti-DR11 of five DR11-reactive antibodies reacted positive, which might indicate residual immunogenic epitopes of DRB1*1101. HLA alleles that are most similar to HLA-DRB1*0340 are DRB1*030501, *0317, *0329 and *1107 with at least four amino acid differences in exon 2. In conclusion, HLA-DRB1*0340 is a new allele with unique properties compared with other known HLA-DRB alleles with regard to antigenicity, T-cell receptor-binding and peptide-binding possibilities.     

Supermotif peptide binding and degeneracy of MHC: peptide recognition in an EBV peptide-specific CTL response with highly restricted TCR usage

We have investigated the presentation and CTL recognition of an HLA A*1101-restricted CTL peptide epitope AVFDRKSDAK (AVF)(3), derived from the EBV nuclear antigen (EBNA) 4, in the context of alleles belonging to the A3-supertype, A*0101, 0301, 1101, 3101, 3301, and 6801. The peptide binds to a A*6801 molecule as efficiently as to A*1101. The A*6801:AVF complex is recognized by some A*1101-restricted AVF- specific CTL clones. However, A*6801-positive (A*6801+) EBV-transformed lymphoblastoid cell lines (LCLs) are not killed by the same effectors. Furthermore, two A*6801+ donors did not mount an AVF-specific CTL response in vitro and lacked detectable AVF-specific effectors. Thus, this epitope is either subdominant, or non-immunogenic in the context of A*6801. These characteristics correlate with low stability of this MHC:peptide complex in living cells. We also demonstrate that a highly conserved AVF-specific TCR that dominates the AVF-specific CTL response in the majority of A*1101+ individuals recognizes the A*6801 molecule as a crossreactive alloantigen. Therefore, deletion of AVF-specific T cells may contribute to the non-immunogenicity or subdominance of the peptide in A*6801+ individuals.     

DRB1*1446 allele variations in the group-specific annealing sites of common amplification primers

A cord blood unit from the Umbilical Cord Bank of Barcelona was initially typed by sequence-based typing (SBT) as DRB1* 0701. However, the subsequent confirmation by sequence-specific oligonucleotide probes (SSOP) revealed an unusual hybridization pattern that did not fit any of the known HLA-DRB1 alleles or allelic combinations. Molecular cloning and sequencing with primers located at introns 1 and 2 provided the definitive identification of a novel DRB1*1446 allele. It was similar to DRB1*1402 at exon 2 except for nucleotide substitutions at codons 10, 11 and 12 (from YST to LRK) that coincide with the 5'-end group specific site for the annealing of common amplification primers used by SBT and sequence-specific primers.     

Beta-hexosaminidase splice site mutation has a high frequency among non-Jewish Tay-Sachs disease carriers from the British Isles.

In the course of defining mutations causing Tay-Sachs disease (TSD) in non-Jewish patients and carriers from the British Isles, we identified a guanine to adenine change (also previously described) in the obligatory GT sequence of the donor splice site at the 5' end of intron 9 of the hexosaminidase alpha peptide gene. Of 24 unrelated mutant chromosomes from 20 non-Jewish subjects (15 TSD carriers, four TSD patients, and one TSD fetus), five had mutations common in the Ashkenazi Jewish community, and 10 had the intron 9 splice site mutation. This is an unexpected result considering the diverse origin of the population of the British Isles. This mutation was not found in 28 control UK subjects or 11 Jewish carriers of known TSD mutations. Before attempting detection of unknown mutations, non-Jewish TSD carriers from the British Isles should be screened for the intron 9 donor splice site mutation as well as those mutations which predominate in the Jewish community.