Molecular structure of RHD negative

• US 9,034,573 B1
• Filed: 10/31/2000
• Issued: 05/19/2015
• Est. Priority Date: 11/02/1999
• Status: Expired due to Fees

• Current Assignee: DRK-Blutspendedienst Baden Württemberg-Hessen gemeinnützige GmbH
• Original Assignee: DRK-Blutspendedienst Baden Württemberg-Hessen gemeinnützige GmbH
• Inventors: Flegel, Willy A., Wagner, Franz F.
• Primary Examiner(s): Strzelecka, Teresa E
• Application Number:
  US10/129,580
• Time in Patent Office: 5,313 Days
• Field of Search: 536/23.1, 536/24.33, 435/6, 435/91.2
• US Class Current: 435/6
• CPC Class Codes: A61P 15/00 Drugs for genital or sexual disorders for disorders of sex hormones A61P5/24; Contraceptives
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: C12Q 2600/172 Haplotypes

Abstract

The RH blood group antigens derive from two genes, RHD and RHCE, that are located at chromosomal position 1p34.1-1p36. In whites, a “cde” haplotype with a deletion of the whole RHD gene occurs with a frequency of about 40%. The relative position of the two RH genes and the location of the RHD deletion was previously unknown. A model for the RH locus was developed using RHD- and RHCE-related nucleotide sequences deposited in nucleotide sequence databases along with PCR and nucleotide sequencing. The open reading frames of both RH genes had opposite orientations. The 3′ ends of the genes faced each other and were separated by about 30,000 base pairs (bp) that contained the SMP1 gene. The RHD gene was flanked by two DNA segments, dubbed Rhesus boxes, that had about 9,000 bp length, 98.6% homology, and identical orientation. The Rhesus box contained the RHD deletion occurring within a stretch of 1,463 bp of identity. A PCR-SSP and a PCR-RFLP for specific detection of the RHD deletion was devised. The molecular structure of the RH gene locus explains mechanisms for generating RHD/RHCE hybrid alleles and the RHD deletion. Specific detection of the RHD negative genotype is now possible. The utility of the RHD PCR is limited by the incomplete knowledge of presumably rare RHD positive alleles in D negative. 1068 serologically RhD-negative samples were checked by PCR-SSP for the presence of RHD specific nucleotide sequences. 48 Samples were positive and were then assigned to specific PCR patterns or distinct RHD alleles. Seven PCR patterns were identified, three of which were not described previously, and four new RHD alleles that were RhD-negative because of nonsense or splice mutations. Another three new haplotypes represented a D_el phenotype. Three samples were mislabeled weak D or partial D. The sensitivity of current RHD PCR methods exceeded routine serology. As the molecular background of D-negative alleles causing false-positive RHD PCR in whites is more heterogeneous than anticipated, improvements in test specificity will critically depend on detecting RhD-negative RHD positive alleles.

Claims

1. An isolated nucleic acid molecule comprising SEQ ID NO:

18 or comprising SEQ ID NO;

20 hybridized to an oligonucleotide that is 12-50 nucleotides in length labelled with a non-nucleotide containing label.

2. The nucleic acid molecule of claim 1 derived from a sample comprising an RHD positive haplotype that is serologically classified RhD negative.

3. The nucleic acid molecule of claim 2, wherein said sample is obtained from a Caucasian.

5. The nucleic acid molecule of claim 1, wherein the label comprises a radioactive marker, or a fluorescent, phosphorescent, chemiluminescent or enzymatic label.

4. A vector comprising SEQ ID NO:

18 or comprising SEQ ID NO;

20.