Hanna Gazda MD, Dana-Farber Cancer Institute, Pediatric Oncology, Rm M615, 44 Binney Street, Boston, MA 02115, USA. E-mail: email@example.com
The genetic basis of Diamond–Blackfan anaemia (DBA), a congenital erythroid hypoplasia that shows marked clinical heterogeneity, remains obscure. However, the fact that nearly one-quarter of patients harbour a variety of mutations in RPS19, a ribosomal protein gene, provides an opportunity to examine whether haplo-insufficiency of RPS19 protein can be demonstrated in certain cases. To that end, we identified 19 of 81 DBA index cases, both familial and sporadic, with RPS19 mutations. We found 14 distinct insertions, deletions, missense, nonsense and splice site mutations in the 19 probands, and studied mutations in 10 patients at the RNA level and in three patients at the protein level. Characterization of the mutations in 10 probands, including six with novel insertions, nonsense and splice site mutations, showed that the abnormal transcript was detectable in nine cases. The RPS19 mRNA and protein in CD34+ bone marrow cells identified haplo-insufficiency in three cases predicted to have one functional allele. Our data support the notion that, in addition to rare DBA patients with the deletion of one allele, the disease in certain other RPS19 mutant patients is because of RPS19 protein haplo-insufficiency.
Diamond–Blackfan anaemia (DBA) is usually characterized by anaemia, absence or insufficiency of erythroid precursors in bone marrow (BM) and normal cellularity of other lineages (Alter & Young, 1998). Although macrocytic anaemia is a prominent feature of DBA, the disease is also characterized by growth retardation and congenital anomalies that are present in approximately half of the patients. Therefore DBA is a broad disorder of development. The genetic basis of DBA remains obscure, but the fact that about one-quarter of the patients harbour a variety of mutations in the ribosomal protein S19 (RPS19) gene provides an opportunity to examine whether haplo-insufficiency of RPS19 protein can be demonstrated in certain cases. To that end, we identified 19 of 81 DBA index cases, both familial and sporadic, with RPS19 mutations (23·5%). We found 14 distinct insertions, deletions, missense, nonsense and splice site mutations of one RPS19 allele in the 19 probands; nine different mutations were novel. Further characterization of the mutations in three patients with novel mutations leading to premature stop codons revealed haplo-insufficiency at the RNA and protein levels in BM CD34+ cells.
Although most DBA cases are sporadic, about 10–25% are familial. In such families, the disease usually behaves as an autosomal dominant disorder; however, some pedigrees seem to be consistent with recessive inheritance. There is excellent evidence that DBA may be a result of at least three different genetic disorders, only one of which, an abnormality of RPS19 on chromosome 19, has been documented and shown to be present in c. 25% of both familial and sporadic cases (Draptchinskaia et al, 1999). Approximately 50% of familial cases are associated with an abnormality on chromosome 8p, but the responsible gene has yet to be identified (Gazda et al, 2001). There is also linkage evidence for a third non-19q, non-8p gene involved in DBA patients (Gazda et al, 2001), some of whom may constitute a clinical subset with cleft palate and microtia (Gripp et al, 2001).
Eighty-one families participated in the study; some of them were included in a previous report (Gazda et al, 2001). Thirty-seven of them were multiplex DBA families; 44 comprised only one affected individual. Informed consent was obtained from all patients and their family members under a protocol at Children's Hospital Boston (Boston, MA, USA) or each country of origin. The diagnosis of DBA in all probands was based on the findings of normochromic anaemia, elevated erythrocyte adenosine deaminase activity (eADA), reticulocytopenia and a low number or lack of erythroid precursors in the bone morrow, often associated with congenital malformations.
Bone marrow CD34+ cells
Bone marrow low-density cells from DBA patients and control individuals were isolated using Histopaque-107 (Sigma-Aldrich Corp., St Louis, MO, USA). Isolated cells were stained with the phycoerythrin cyanin 5 anti-human CD34 antibody (BD Biosciences Pharmingen, San Diego, CA, USA) and cells expressing CD34 antigen were sorted using the ALTRA HyPeSort System (Beckman Coulter, Miami, FL, USA). For protein extraction, 3 × 105 cells were washed, boiled for 10 min in sample buffer and stored at −20°C. Protein extracts were quantified by spectrophotometer using Bio-Rad DC Protein Assay (reagents A, B, S) (Bio-Rad Laboratories, Hercules, CA, USA).
Peripheral blood mononuclear cells
Peripheral blood mononuclear cells (MNC) from DBA patients and control individuals were isolated using Histopaque-107 (Sigma-Aldrich Corp.). For protein extraction, 2 × 106 cells were processed as described above.
Generation of anti-RPS19 antiserum
The glutathione S-transferase (GST) fusion protein strategy was adopted to generate rabbit polyclonal antibodies to RPS19, as described (Kaelin et al, 1991; Frangioni & Neel, 1993; Yamashita et al, 1994). An RPS19-specific immune antiserum was affinity purified over an AminoLink Plus column (Pierce, Rockford, IL, USA) loaded with GST protein and subsequently over an AminoLink Plus column loaded with GST-RPS19.
DNA isolation and polymerase chain reaction
Genomic DNA (gDNA) was isolated from whole blood by standard methods (Engle et al, 1994). The DNA samples from 81 probands enrolled in the study and from 50 control individuals were amplified by polymerase chain reaction (PCR). Five pairs of primers were used to amplify fragments that contained the 5′UTR and the six exons of the RPS19 gene. The PCR products were purified with a PCR purification kit (Qiagen, Valencia, CA, USA).
RPS19 gDNA sequencing
The PCR primers and additional internal primers were used for fluorescent DNA sequencing as previously described (Draptchinskaia et al, 1999). All samples were sequenced using forward and reverse primers. Duplicate, independent PCR products were sequenced to confirm the observed nucleotide changes in the probands. DNA from 50 control individuals, i.e. 100 chromosomes, was sequenced to determine if the sequence variation was a pathogenic mutation or a polymorphism. DNA samples from all available family members were sequenced to determine whether the mutation co-segregated with the DBA phenotype within the pedigree.
RNA isolation and reverse transcription-polymerase chain reaction
Total RNA was isolated from BM CD34+ cells and MNC (described above) from DBA patients and control individuals using an RNA isolation kit (Qiagen) according to the manufacturer's instructions. The mRNA fractions from the MNC of ten patients and control individuals were reverse transcribed into cDNA and amplified by One-step reverse transcription-polymerase chain reaction (RT-PCR) kit (Qiagen) using primers that flanked the beginning and the end of the coding region. Control reactions without the inclusion of reverse transcriptase were utilized to exclude contamination of the cDNA by gDNA. The size of the RT-PCR products was confirmed on 1·3% agarose gels. The RT-PCR products were purified, sequenced and analysed as described above.
Quantitative real-time RT-PCR
RPS19 transcripts from BM CD34+ cells and MNC from DBA patients and control individuals were quantified by real-time RT-PCR (Applied Biosystems, Foster City, CA, USA) with an ‘Assays-by-Design’ gene expression kit with primers and probe designed in exon 4. RNA from BM CD34+ cells (c. 200 ng) and from peripheral blood cells (1 μg) from DBA patients and normal individuals was reverse-transcribed to cDNA (TaqMan Reverse Transcription Reagents; Applied Biosystems). Parallel control reactions without reverse transcriptase were run to exclude contamination of the RNA by gDNA and amplification of gDNA. The obtained cDNAs were amplified in duplicate in a real-time quantitative PCR reaction using the TaqMan Universal PCR Master Mix, the recommended TaqMan protocol and the ABI 7700 Sequence Detection System (Applied Biosystems). Control reactions with the human GAPDH gene (‘Assays-by-Demand’; Applied Biosystems) were run in the same tubes with RPS19 as an endogenous reference for the RPS19 target gene. The outcome of each amplification was calculated with comparative methods according to the manufacturer's protocol. This procedure enabled calculations of the RPS19 expression level and fold changes between DBA and normal samples normalized to GAPDH in each RNA sample.
For Western blot analysis 70 µg protein lysate from BM CD34+ cells and 100 Bg of protein lysate from MNC were electrophoresed on 15% sodium dodecyl sulphate-polyacrylamide or 15% tricin gels and transferred to nitrocellulose membranes by electroblotting. The RPS19 protein was detected with the rabbit polyclonal RPS19 antibody and anti-rabbit IgG horseradish peroxidase-conjugated antibodies (Upstate Biotechnology; Lake Placid, NY, USA). Proteins were visualized by enhanced chemiluminescence (ECL; GE Healthcare, Piscataway, NJ, USA). Equivalent loading of lanes was determined by Ponceau S staining and staining of the membrane with β-tubulin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Quantification of the RPS19 protein levels normalized to β-tubulin was performed with the program quantity one version 4.2.1 (Bio-Rad Laboratories, Hercules, CA, USA), on Image Station 440 (Kodak DS, New Haven, CT, USA).
RPS19 gene mutations in 81 unrelated probands
To determine RPS19 mutations we sequenced the gDNA of 81 unrelated DBA probands, both familial and sporadic cases. We also analysed 74 family members of the probands in whom we found a mutation. Pathogenic mutations were defined as a sequence change predicted or shown to alter the protein product and not observed in 50 control individuals, i.e. 100 normal chromosomes. One nonsense mutation found in two unrelated patients (P-12 and P-13) caused a premature stop codon in exon 2. In two cases (P-14 and P-15), insertions of two and one nucleotides resulted in frameshifts and premature stop codons. In addition, we found 11 other mutations in one of the RPS19 alleles in 15 index cases. In total, we found 14 different mutations in 19 of 81 probands (23·5%); 10 probands were familial cases and nine were sporadic. These mutations are detailed in Table I and illustrated in Fig 1.
Table I. RPS19 gene mutation in familial and sporadic DBA patients.
Frameshift at codon 84; stop codon 7 amino acids behind coding region
Frameshift at codon 115; stop at 123
One proband was described¶
Splice site mutations
Acceptor splice site IVS3-2g>a agCTTC>aaCTTC
Deletion 3 amino acids: AlaSerThe
Donor splice site IVS4 +1g>a ATGGgt>ATGGat
Deleted exon 4; frameshift at exon 3/exon 5; stop at codon 124 (wt)
Truncated protein comprising 61 amino acids
Using a one-step RT-PCR assay we amplified and sequenced cDNAs from the peripheral blood of 10 DBA patients with RPS19 mutations found in gDNA. The missense and nonsense mutations in five patients, including P-12, as well as insertions of a single nucleotide in two patients (P-15 and P-16) were confirmed at the cDNA level. In patient P-14 the cDNA sequence detected only a normal transcript in three replicate experiments. The mutation caused a shift of the reading frame at codon 13 and a stop at codon 29. Interestingly, the amplified cDNA in P-19 revealed two transcripts, wild type (wt) (c. 480 bp) and mutant (c. 300 bp) on an agarose gel (data not shown). The bands were cut, purified and sequenced. The cDNA sequence revealed the aberrant form of RNA splicing with deletion of the whole exon 4, shift of the reading frame and premature termination codon [at codon 124 (wt)]. The cDNA changes are detailed in Table I.
Quantitative real-time RT-PCR
We tested the quantity of RPS19 mRNA normalized to GAPDH level in BM CD34+ cells in three patients (P-12, P-14 and P-15) with premature termination codons and in four control individuals. We found a two to fourfold decrease of the RPS19 mRNA in BM CD34+ cells in all three patients compared to control individuals (Fig 2A and B). We performed the same experiments using RNA from MNC from five normal individuals and from six patients; four of them had premature stop codons (P-12, P-14, P-15 and P-19), while two other patients had missense (P-10) or splice site (P-18) mutations. The experiments revealed a two to fourfold decrease of the mRNA in the three patients with premature termination codons (P-12, P-14 and P-15), while patients P10, P-18 and P-19 did not show reduction of the RPS19 mRNA compared with control individuals. Most likely, the abnormal transcripts in these patients and the spliced exon 4 in patient P-19 were also detected by the quantitative real-time RT-PCR (Fig 2C and D). Real-time RT-PCR experiments with MNC RNA from patient P-12 were performed separately (the blood sample from this patient was collected after other MNC experiments were completed) and showed the same reduction of the RPS19 mRNA normalized to GAPDH mRNA as in the CD34+ BM cells (Fig 2C1 and D1).
RPS19 protein analysis in BM CD34+ cells and in peripheral blood MNC
Western blot analysis of RPS19 protein expression in BM CD34+ cells was performed in two patients, P-12 and P-15 with premature termination codons and in four control individuals. RPS19 protein expression was normalized to β-tubulin expression by quantitative densitometry. We found three and twofold reductions of the RPS19 protein in BM CD34+ cells in patients P-12 and P-15, respectively, compared with normal individuals (Fig 3A). Interestingly, there was no significant difference in the expression of the RPS19 protein in MNC between these patients or patient P-19 and control individuals (Fig 3B). We were not able to visualize the putative truncated protein in protein lysates from these patients. The data were confirmed in four independent experiments.
Clinical heterogeneity in DBA families as a result of RPS19 mutations
Direct sequencing of gDNA in family members from 17 of 19 index cases with RPS19 mutations was performed to assess possible correlations between phenotypes and genotypes. Healthy family members of sporadic cases were either not available for study or lacked RPS19 mutations.
In the multiplex families, a full correlation between the ‘classical’ DBA features and the RPS19 mutations was found in two of 10 pedigrees (P-1 and P-4) (Table I). In both families, mutations were found in affected individuals who presented with severe anaemia, macrocytosis and a high level of eADA. All patients were dependent on small doses of steroids after the first course of steroid treatment. Asymptomatic family members did not share the mutations. In the other eight families, both with known and novel mutations, no apparent phenotype–genotype correlation was observed. The family P-7 is an example of a large pedigree with tremendous clinical heterogeneity. The 185G>A transition was detected in seven individuals who were screened for RPS19 mutation. Six individuals presented with severe macrocytic anaemia, elevated eADA, short stature (<3–5 percentile) and a good response to steroid treatment. In addition, the mutation was found in an apparently healthy individual who only had transient macrocytic anaemia during pregnancy and elevated eADA. The father of the affected half-siblings, who was not available for study and was most probably a gene carrier, was a ‘pale’ child never treated for anaemia; he was noted to be short (5 percentile). Clinical features of the family P-7 were described elsewhere (Viskochil et al, 1990) and the genetic status of the proband was mentioned previously (Gazda et al, 2001).
Family P-2 was particularly interesting (Cmejla et al, 2000). There were two affected sisters: one is currently in remission, the other has transfusion-dependent anaemia. Neither parent is affected in any apparent way. This family phenotype suggested autosomal recessive inheritance. Direct sequencing revealed a missense mutation of one RPS19 allele in exon 3, 167G>A, resulting in the change of arginine to glutamine at codon 56 in both affected sisters but not in their parents. Extensive genotyping confirmed the parenthood of both mother and father (data not shown). The finding most probably suggests germ line mutation in one of the parents.
Although haplo-insufficiency has been suggested as the mechanism underlying the pathogenesis of DBA, in no case (to our knowledge) has protein evidence supported this speculation; two patients with disruption or loss of the entire RPS19 allele showed a reduced level of RNA (Hamaguchi et al, 2002). Evidence provided here in three non-deletion patients (P-12, P-14 and P-15) with either missense or insertion/frameshift mutations that result in premature stop codons showed that both protein and/or RNA were reduced in CD34+ cells. These data strongly support the notion that haplo-insufficiency underlies the pathogenesis of DBA, not only in those ‘obligatory’ haplo-insufficient patients with deletion or disruption of an entire allele (Gustavsson et al, 1998; Hamaguchi et al, 2002; Campagnoli et al, 2004), but also in other patients. Whether loss of function is the mechanism in all RPS19 mutant patients is uncertain, but the broad distribution across all coding exons of insertions/deletion, splice site and nonsense mutations, together with the extreme clinical heterogeneity of DBA, as illustrated by the family of P-7, support this conclusion. Conditions with a broad variety of mutations and clinical heterogeneity within families are usually the result of haplo-insufficiency influenced by genetic and/or environmental modulating factors. The Waardenburg syndrome type I is an excellent example of loss of function caused by different mutations in the PAX3 gene (Baldwin et al, 1995; DeStefano et al, 1998), as is Williams syndrome and the variable output of the elastin gene (Ewart et al, 1993, 1994; Morris & Mervis, 2000). Interestingly, in Waardenburg syndrome mutations that disrupt the structure of the PAX3 gene are scattered over the first six exons of the gene, while missense mutations cluster in two important functional domains, consistent with loss of function in both contexts. DBA may be analogous, in that patients with deletion of an entire allele, and most likely the patients presented here with nonsense mutations or premature stop codons, have loss of function through haplo-insufficiency, while those with missense mutations, a majority of which appear to cluster in a ‘mutational hotspot’, are likely to have loss of function through mutations in an important functional domain of the gene.
We did not detect the abnormal transcript in peripheral blood in patient P-14. The insertion of two nucleotides in exon 2 in this patient caused a frameshift at codon 13 and premature termination at codon 29, i.e. in the first 20% of the coding region length. The most likely explanation for this finding is that the premature termination codon triggers nonsense-mediated mRNA decay, which rapidly degrades the abnormal mRNA (Lykke-Andersen et al, 2001; Maquat & Carmichael, 2001). However, sequencing of RPS19 RT-PCR products from MNC of patients P-12, P-15 and P-19 revealed both the wt and abnormal transcripts. Translation terminated more than 50–55 nt upstream of the last exon–exon junction in P-12 and P-15, suggesting that the aberrant mRNA in these patients is also subject to decay, and that only one transcript is translated (Kim et al, 2001). In P-19, the premature termination at codon 124 occurred <50 nt upstream of the last exon–exon junction, and the abnormal transcript was most probably spared from degradation (Cheng et al, 1994; Carter et al, 1996; Kim et al, 2001). The results of quantitative real-time RT-PCR supported this interpretation. The RPS19 mRNAs from BM CD34+ and from MNC were reduced by two to fourfold in all three patients (P-12, P-14 and P-15) compared with control individuals. These results also imply that haplo-insufficiency is the cause of the DBA in these patients. The quantitative real-time RT-PCR assay revealed the same amount of the RPS19 mRNA in MNC in P-19 and in control individuals. The patients with a missense or splice site mutation (P-10 and P-18) did not show reduction of the RPS19 mRNA either. It is most likely that the abnormal transcripts in these patients and the spliced exon 4 in patient P-19 were also detected by the quantitative real-time RT-PCR. To further investigate the abnormal transcript in P-19 we used a second set of real-time RT-PCR primers and probe located at the 5′ end of the RPS19 coding region. Again we found the same amount of the RPS19 mRNA in MNC in P-19 and in control individuals, which confirms that the abnormal transcript in this patient is spared from degradation. To confirm at the protein level that haplo-insufficiency is a cause of DBA in patients with one functional allele we performed Western blot analyses of RPS19 protein expression in BM CD34+ cells in P-12 and P-15. We found three and twofold decreases of the RPS19 protein in P-12 and P-15 (Fig 3A).
In contrast to CD34+ cells, expression of the RPS19 protein in MNC in patients P-12, P-14 and P-15, did not differ from that of control individuals (Fig 3B). There are two possibilities. First, it is possible that RPS19 regulation is different in MNC, which are unaffected in DBA, compared with CD34+ cells, a population that contains the erythroid progenitors. There may be variability of expression of the normal allele in MNC; those with low levels may die while those with normal levels may be selected for survival. It is likely that erythroid progenitors lack any compensatory capability and are therefore susceptible to apoptotic death (Perdahl et al, 1994) because of RPS19 insufficiency. Why such insufficiency promotes the apoptotic pathway in human erythroid progenitors remains an unanswered question. Secondly, there is a maturation-related decrease in RPS19 expression in erythroid cells with the highest levels in proerythroblasts (Da Costa et al, 2003), and it is possible that full expression from both alleles is required in early erythroid cells; however, lower levels of RPS19 protein may suffice in the more mature MNC, either the output of one functional allele or a decreased contribution from each of two normal alleles. The data strongly suggest that replacement of RPS19 in erythroid progenitors by transfection of stem cells with a vector containing the wild type gene should improve haematopoiesis in these patients. Although in vitro attempts to date have only partially corrected the defect (Hamaguchi et al, 2002, 2003), such efforts should certainly continue.
In conclusion, it is likely that the phenotype of DBA because of nonsense mutations, insertions, deletions and splice site mutations with frameshift, premature stop codon and mRNA decay in RPS19 is caused by haplo-insufficiency, and the marked clinical heterogeneity depends on the influence of additional genetic and/or environmental modulators.
The authors acknowledge Giesela Schluh for the administrative work for the DBA patients in Germany. We gratefully acknowledge the DBA patients and their family members who participated in this study.
This work was supported by grants from the NIH SCOR HL99-021, RO1 HL64775, Diamond–Blackfan Anemia Foundation and German Federal Ministry of Education and Research (BMBF).