A four base pair insertion in exon 1 of the RPS19 gene is a common polymorphism in African‐Americans (2006)

Diamond–Blackfan anaemia (DBA) (On-line Mendelian Inheritance in Man No. 205900) is a pure red cell aplasia that usually presents in early infancy, with little or no suppression of other blood cell lineages. In about one quarter of cases DBA is familial and it is associated with mutations in the RPS19 gene, which encodes small subunit ribosomal protein 19 (RPS19), in about one quarter of cases, whether familial or not. As yet, no other DBA genes are known. More than 60 different mutations in the RPS19 gene have now been described. These include missense, nonsense and frameshift mutations, as well as splice site defects and large gene deletions. One of several recurring mutations is a 4-bp insertion in the non-coding portion of exon 1 (Willig et al, 1999; Ramenghi et al, 2000; Campagnoli et al, 2004; Orfali et al, 2004), which has been found in five DBA patients. Owing to polymorphisms and the fact that the insertion site is in the sequence AA, this sequence alteration has been referred to historically in different ways (Willig et al, 1999; Ramenghi et al, 2000; Orfali et al, 2004). In an attempt to clarify this, we have referred to the insertion as an insertion of the sequence GCCA at −631 in the RPS19 gene, where the base before the A of the initiation ATG is assigned the position −1 and the reference sequence is the latest version of the human genome found in the ENSEMBL database at the Sanger Institute (http://www.sanger.ac.uk). The sequence 5′-GGGGACGAA CTTTCGCCC-3′ becomes 5′-GGGGACGAA GCCA CTTTCGCCC-3′. We report an analysis of panels of control individuals of different ethnic origins. It was found that the −631 insertion was polymorphic in African-Americans with an allele frequency of 0·09. Healthy volunteers or patients who presented for reasons other than bone marrow failure were screened for the insertion by polymerase chain reaction (PCR). The local ethics committee approved the study. None of the 80 Caucasian individuals that were screened was heterozygous for the 4-bp insertion in exon 1 of RPS19 gene. Of 186 African-American samples tested, 33 were heterozygous for the insertion in exon 1 and one was homozygous (Fig 1). That the insertion detected by PCR was the −631 GCCA insertion was confirmed in five cases by sequencing. Thus, in our African-American sample, the gene frequency for the allele with the −631 insertion was 0·09. Such a gene frequency rules out the possibility that heterozygosity for the −631 insertion is a cause of DBA, which has a frequency of <10 per million. (A) A representative polyacrylamide gel showing polymerase chain reaction amplification products from the segment of RPS19 containing the −631 insertion. All the samples were from African-Americans. The gel shows that two samples are heterozygous for the −631 insertion. (B) As in (A) showing homozygous normal (lanes 1 and 4), heterozygous (lane 2), and the one homozygous sample found in this study (lane 3). There are several other observations that support this conclusion. First, the insertion occurs in part of the gene with no clear function and, in in vitro experiments, the insertion had no effect on the expression of RPS19 (Da Costa et al, 2003). Secondly, the insertion has been reported in DBA patients with another abnormality in the RPS19 gene (Willig et al, 1999). Our finding that the −631 insertion is a polymorphism which is important for the correct diagnosis of DBA patients and their families, particularly those within the African-American population. DBA is one of the diseases for which prenatal diagnosis and pre-implantation diagnosis is available and practiced (Dyer, 2006). In addition, haematopoietic stem cell transplantation is the only potential cure. For this, family members are tested for the mutation present in the proband and excluded as donors if they carry the same sequence alteration. Our study showed that the −631 insertion, which has been identified in multiple individuals with DBA, is a polymorphism in the healthy African-American population with a frequency of 9%. Incorrect assignment of the −631 insertion as a pathogenic mutation could lead to the abortion of a normal fetus or exclusion of a potential donor. This work was supported by the Daniella Maria Arturi Foundation and by the NIH NCI CA105312. We would like to thank Jennifer Ivanovitch and the Hereditary Cancer Core of the Siteman Cancer Center for supplying the cancer-free controls The Siteman Cancer Center is supported in part by an NCI Cancer Center Support Grant no. P30 CA91842.