Identification of a new HLA-DRB5 allele, DRB5*0112, by routine PCR-SSP

We describe the identification of a new HLA-DRB5 allele found in two members of a British Caucasoid family. Genomic analysis of exon 2 revealed a novel sequence. The name DRB5*0112 has been assigned to this allele by the WHO Nomenclature Committee, and the EMBL sequence database accession number for this is AJ427352. DRB5*0112 has several unique sequence features when compared to other DRB5 alleles, and comparison with all known DRB1/3/4/5 alleles indicates this to have arisen as a result of intraexonic recombination between a DRB5-containing haplotype and one carrying DRB1*09.     

Comparison of two HLA-DRB high resolution microtiter plate reverse hybridization typing methods: advantage of a codon-86 valine or glycine PCR segregation

Abstract: Two rapid, nonisotopic, high-resolution HLA-DRB typing methods have been developed for DRB1, DRB3, DRB4 and DRB5 alleles. These methods are based on a single procedure consisting of the reverse hybridization of biotinylated amplicons to oligonucleotide probes that are covalently attached to a microtiter plate. Detection is by an enzymatic reaction with a fluorescent substrate. The 1 Generic amplification (1GA) method amplifies all HLA-DRB alleles in the same reaction mix. The 2 Allelic Subset amplification (2SA) method uses two distinct amplification reactions that distributes all DRB alleles into two equal-size subsets, according to the codon 86 Gly or Val polymorphism; this adds an extra discrimination level to the typing. 108 samples were typed using the 1GA and the 2SA methods and no discrepancies were found. Typing indeterminations due to overlapping probe combinations were compared; it was found that the 2SA method, with the extra discrimination level at the PCR step, greatly improved resolution.     

Identification of a new HLA-DRB4 allele (DRB4*01033) by PCR-SSP and direct sequencing

In this report, we describe the identification of a novel DRB4*01 allele, DRB4*01033, found in two Spanish Caucasian individuals. The new allele was detected during routine HLA typing by an unusual pattern of amplification obtained by polymerase chain reaction using sequence-specific primers (PCR-SSP) that did not match with any of the previously described DRB4 alleles. In order to establish the polymorphism responsible for this pattern exons 2 and 3 of the DRB4 locus were amplified and directly sequenced. The new DRB4*01 allele is identical to DRB4*0103101 except for a single nucleotide substitution in codon 78 (TAC-->TAT). This nucleotide change does not cause an amino acid change as both triplets code for a tyrosine.     

High resolution HLA-C typing by PCR-SSP: identification of allelic frequencies and linkage disequilibria in 604 unrelated random UK Caucasoids and a comparison with serology

Recent evidence indicates that HLA-C molecules are biologically relevant by eliciting T-cell responses and exerting control over NK cell function. In addition, HLA-C is associated with susceptibility to various diseases, notably psoriasis vulgaris. Clarification of the full biological roles for HLA-C has however proved difficult because detection of HLA-C antigens by complement mediated cytotoxicity using alloantisera is inefficient. Up to 50% of individuals in every race have serologically undetectable HLA-C locus antigens due to a combination of relatively low expression, lack of serological reagents and a lack of information about the distribution of the HLA-C blank alleles. Recently, amplification of DNA using sequence-specific primers (PCR-SSP) has proved a reliable, accurate and rapid method for medium resolution HLA-C typing. We have now developed high resolution HLA-C typing by PCR-SSP utilizing allele and group-specific PCR-SSP reactions which can identify all HLA-C alleles (except non-coding change alleles) in most heterozygous combinations. Using this system we have typed 604 unrelated United Kingdom Caucasoids to generate accurate frequency and linkage disequilibrium data. To assess the validity of serology for HLA-C, PCR-SSP typings for 527 out of the 604 individuals were compared to serology. We find that the frequency of many HLA-C antigens has been underestimated by serology and some antigens such as Cw6 are consistently assigned incorrectly by serology. The overall discrepancy rate between serology and SSP was high at 37% (195/527). High-resolution HLA-C typing of 112 International Histocompatibility Workshop cell lines has also been performed.     

High resolution HLA-C typing by PCR-SSP: identification of allelic frequencies and linkage disequilibria in 604 unrelated random UK Caucasoids and a comparison with serology

Recent evidence indicates that HLA-C molecules are biologically relevant by eliciting T-cell responses and exerting control over NK cell function. In addition, HLA-C is associated with susceptibility to various diseases, notably psoriasis vulgaris. Clarification of the full biological roles for HLA-C has however proved difficult because detection of HLA-C antigens by complement mediated cytotoxicity using alloantisera is inefficient. Up to 50% of individuals in every race have serologically undetectable HLA-C locus antigens due to a combination of relatively low expression, lack of serological reagents and a lack of information about the distribution of the HLA-C blank alleles. Recently, amplification of DNA using sequence-specific primers (PCR-SSP) has proved a reliable, accurate and rapid method for medium resolution HLA-C typing. We have now developed high resolution HLA-C typing by PCR-SSP utilizing allele and group-specific PCR-SSP reactions which can identify all HLA-C alleles (except non-coding change alleles) in most heterozygous combinations. Using this system we have typed 604 unrelated United Kingdom Caucasoids to generate accurate frequency and linkage disequilibrium data. To assess the validity of serology for HLA-C, PCR-SSP typings for 527 out of the 604 individuals were compared to serology. We find that the frequency of many HLA-C antigens has been underestimated by serology and some antigens such as Cw6 are consistently assigned incorrectly by serology. The overall discrepancy rate between serology and SSP was high at 37% (195/527). High-resolution HLA-C typing of 112 International Histocompatibility Workshop cell lines has also been performed     

'Silent' carriage of two familial Mediterranean fever gene mutations in large families with only a single identified patient

The presence of two mutations in the familial Mediterranean fever gene, without overt familial Mediterranean fever (FMF), designated as phenotype III, predisposes to developing 'silent' AA amyloidosis, recognized as phenotype II, due to the absence of medical supervision and colchicine prophylaxis. We sought to determine the prevalence of phenotype III in large families with only one subject affected with FMF, in order to assess the population at risk for transformation to phenotype II. A total of seven large families were recruited for the study. Siblings were screened for MEFV mutations and underwent a clinical interview to assess for unrecognized FMF manifestations. Phenotype III, most commonly associated with a V726A/E148Q genotype, was detected in 10% of siblings of index cases from informative families, corresponding to a 10-fold increase in comparison to the expected rate in the general population (p < 0.01). Unnoticed 'FMF-like' manifestations were detected among two siblings in the five families in which the index case was heterozygous, but in none of the siblings of the homozygous index cases. The enrichment for phenotype III and detection of occult FMF in large families, in which only a single member is afflicted with FMF, mandates routine clinical evaluation and genetic screening of siblings.     

Determination of gene frequencies for two common haemochromatosis mutations in the Danish population by a novel polymerase chain reaction with sequence-specific primers

Abstract: Hereditary haemochromatosis (HH), a condition of abnormal iron metabolism which leads to iron overload and organ damage, previously known as bronze diabetes or idiopathic haemochromatosis, is the most common disease-producing genetic disorder among Europeans. Two mutations, C282Y and H63D, are described for the candidate gene, HFE, reported as being responsible for the disease. Since molecular testing of these mutations will be of value in early diagnosis of haemochromatosis, the aim of this study was to develop a simple, fast and inexpensive technique for the determination of the polymorphism in the HFE gene on a large scale. We designed sequence-specific primers for polymerase chain reaction (PCR-SSP) and tested 200 randomly selected healthy Danes and found the result completely comparable to results obtained by a previously described method, PCR-RFLP. The gene frequencies in the Danish population are similar to reported results for the White population, with a frequency of 0.068 for the C282Y mutation and a frequency of 0.128 for the H63D mutation.     

Identification of two new alleles in a single Korean individual,HLA-B*1568 and HLA-DRB1*1208

We have identified a new HLA-B*15 allele and a new HLA-DRB1*12 allele, named B*1568 and DRB1*1208, respectively. The alleles were identified using a combination of sequence specific primers, reverse line sequence specific oligonucleotide probing and sequence-based typing. Both alleles were identified in a single individual of Korean origin. HLA-B*1568 appears to be an HLA-B*4801/B*1507 hybrid combining the exon 2 sequence of B*4801 and the exon 3 and 4 sequences of B*1507. Exon 2 of DRB1*1208 was most similar to DRB1*1201 or 1206, with a single mismatch at nucleotide position 165 (A to C). At the protein level, this substitution results in a phenylalanine substitution at position 26 that creates an identical amino acid sequence to DRB3*0202 between amino acid positions 17 and 36.     

Identification of a new DQB1 allele that appears to have been generated by an interallelic sequence exchange

The study reports the molecular characterization of a new DQB1 variant initially detected by unusual sequence-specific oligonucleotide (SSO) hybridization patterns in one Caucasoid individual. This new allele is identical to DQB1*0501 except for two silent nucleotide substitutions at codons 49 (GCA-->GCG) and 77 (AGG-->AGA). Compared with DQB1*0502 it differs in three nucleotides at codon 57 changing AGC (encoding Ser) to GTT (encoding Val). Considering the paternal genotype, it appears this new allele might have been generated by an interallelic sequence exchange between the two paternal DQB1 alleles.     

一例多位点突变导致的罕见B(A)型的血清学及分子遗传学研究

目的探讨1例ABO血型血清学正、反定型不符的血样标本的分子遗传学基础。方法采用试管法对此例血样进行ABO血型正、反定型,应用直接测序及单倍型测序的方法确定其基因型。结果此例样本血型血清学结果显示,其正定型为AwB型,反定型为B型;直接测序及单倍型测序最终结果显示,其中一条等位基因为O01,另一条等位基因与A101标准序列相比,存在c.297A>G、c.657C>T、c.796C>A、c.803G>C及c.930G>A碱基突变。结论经基因测序证实,此例血清学定型困难的样本基因型为O01/B(A)new型,此突变类型的B(A)型既往未见报道。     

Sequencing of a new HLA-DR4 allele with an unusual residue at position 88 that does not affect T cell allo recognition.

It is rather common to encounter new HLA alleles identified by unorthodox patterns observed during low resolution typing performed with sequence specific oligonucleotide probes (SSOP). One of the best examples is locus DRB1, where allelic subtypes are characterized by a combination of a limited number of residues located in three hypervariable regions of exon 2. HLA-DR oligotyping analysis of a female caucasoïd bone marrow donor of the Lyon registry led to the identification of an individual that typed as DRB1^* 11, DRB3^* 02, DRB4^* 01, DQB1^* 0301-0302. This donor was however typed by serology as DR11 DR4, DR52 DR53, DQ7 DQ8. PCR-SSP typing for DR4 subtype revealed an amplification pattern typical for DRB1^* 0404. After PCR amplification of genomic DNA and sequencing the entire exon 2, a new DRB1 allele was identified : DRB1^* 04var that is identical to DRB1^* 0404, except for one nucleotide at codon 88 resulting in a Ser ==> Arg exchange. This mutation had prevented amplification with the DR generic primers. The donor inherited this new DRB1^* 04var from her mother who presented the same molecular characteristics. The complete SSOP typing of the implicated haplotype is : A^*68011, Cw^*0704, B^*4402, DRB1^* 04var, DRB4^* 0103, DQA^* 0301, DQB1^* 0302, DPB1^* 0601.

Cellular typing by three HTCs-DRB1^*0404/DW14 from the 9th Workshop showed that this DRB1^* 04var typed exactly like a DW14 cell (the S.R.R. being 40%, 34%, 36%, respectively). This suggests that residue 88 does not affect T cell recognition possibly due to its location at the far end of the alpha helix flanking the peptide binding groove.     

Exome sequencing detection of two untranslated GFPT1 mutations in a family with limb‐girdle myasthenia

The term ‘limb‐girdle myasthenia’ (LGM) was first used to describe three siblings with proximal limb weakness without oculobulbar involvement, but with EMG decrement and responsiveness to anticholinesterase medication. We report here that exome sequencing in the proband of this family revealed several sequence variations in genes linked to proximal limb weakness. However, the only mutations that cosegregated with disease were an intronic IVS7‐8A>G mutation and the previously reported 3′‐UTR c.*22C>A mutation in GFPT1, a gene linked to LGM. A minigene assay showed that IVS7‐8A>G activates an alternative splice acceptor that results in retention of the last seven nucleotides of intron 7 and a frameshift leading to a termination codon 13 nucleotides downstream from the new splice site. An anconeus muscle biopsy revealed mild reduction of the axon terminal size and postsynaptic fold simplification. The amplitudes of miniature endplate potentials and quantal release were also diminished. The DNA of the mildly affected father of the proband showed only the intronic mutation along with sequence variations in other genes potentially relevant to LGM. Thus, this study performed in the family originally described with LGM showed two GFPT1 untranslated mutations, which may cause disease by reducing GFPT1 expression and ultimately impairing protein glycosylation.