SummaryThe 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 haploinsufficiency 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 anemia (DBA) is a congenital red cell aplasia with marked clinical heterogeneity, an increased risk of malignancy and mutations in ribosomal protein (RP) S19 in 25% of probands. To identify other gene(s) mutated in DBA and investigate their expression and function, we performed a genome-wide screen using a 10,000 single nucleotide polymorphism mapping set (Affymetrix) on a large family comprising 10 informative meioses. We found linkage of the DBA phenotype to regions on chromosome 8q, 10 and 6. The RP gene RPS24, is located in the linked region on chromosome 10; we sequenced exons, intron-exon boundaries and the promoter regions in this family, and found a nonsense mutation (316C>T) in exon 4 of RPS24 in five affected individuals, and a wild type sequence in five unaffected family members. This mutation causes the change of Gln106STOP and is predicted to result in formation of a truncated RPS24 protein. Subsequently, we sequenced DNA from 215 unrelated DBA probands, 30 with RPS19 mutations and 185 without. We found another nonsense mutation in exon 2 in a sporadic case, and a splice site deletion resulting in skipped exon 2 in another proband and in his father; over 200 control individuals did not have any of the above sequence changes, indicating that they are pathogenic mutations. To explore the normal role of RPS24 and consider how its dysfunction might result in DBA we performed real time RT-PCR (rt-PCR) and western blotting experiments on 20 normal human tissues and on lymphoblastoid cell lines from diseased and control individuals. Interestingly, rt-PCR of total human RPS24 and RPS19 mRNA revealed a tissue-specific variation in expression level. We found co-ordinate expression of both genes in the majority of studied tissues. Lymphoblastoid cell lines from both probands with nonsense mutations showed a reduced level of RPS24 mRNA, suggesting degradation of mutated transcripts due to nonsense mediated decay, while the RPS19 mRNA level in these patients was normal or elevated. Western blot experiments revealed a reduction of RPS24 protein in lymphoblastoid cell lines from all three mutated probands compared to control samples. Interestingly, co-ordinate expression of RPS24 and RPS19 protein was found in these patients as well as in other patients with RPS19 mutations or without any mutations, suggesting co-regulation of RP expression. To determine whether recruitment of mRNA to polysomes was impaired in DBA patients, we separated lymphoblast cell line lysates from nine diseased and four control individuals on sucrose gradients. We did not detect any significant difference in the RNA ratio of polysome-bound/free ribosomal subunits between diseased and control samples (p<0.3). It is likely that lymphoblasts, which are not defective in DBA, have sufficient ribosomes to mask an abnormality in translation. In summary, our results suggest that in addition to RPS24 and RPS19, which are mutated in ~ 27% of index cases, other DBA genes are also RP genes or genes involved in ribosome biogenesis or translation, and reinforce the notion that DBA is a ribosomal disease. This study opens new avenues for studying and understanding the pathogenesis of DBA.

A mutation in the GIRK2 inwardly rectifying K ÷ channel was mapped recently to the region of mouse chromosome 16 containing the wv gene and shown to occur in mutant but not in wild-type mice. We demonstrate tight linkage of the Girk2 mutation to the wv phenotype and refine the localization of the weaver (wv) gene on recombinational and physical maps. This linkage between Girk2 and ~ has existed since at least 1988 in descendants of the original mutation maintained in CS7BLI6 animals. Giric2. is shown to be transcribed in brain before the first recognized manifestation of the wv phenotype and in cultures of granule cells (GCs} isolated from cerebellum at postnatal day 8. Wild-type GCs grown in this culture system display an important developmental property--the ability to extend neurites. However, no inwardly rectifying K + current is detected in GCs cultured from either wv/~ or +/+ cerebellum under a variety of conditions that activate related channels in other tissues. This suggests that if the Gir~ mutation is responsible for the wv phenotype, it does not act by altering these electrical properties of developing GCs.The weaver (wv) mutation in mice was first characterized in 1973 as a recessive mutation resulting in hypoplasia of the cerebellar internal granule layer (IGL) (Rakic and Sidman 1973). As a consequence, wv/wv mice display severe ataxia. Heterozygous (+/wv) individuals are not ataxic, but develop a significantly smaller cerebellum than do control animals. The wv gene maps to a region of mouse chromosome 16 (Chr 16) that is highly conserved with human chromosome 21 (HSA21), suggesting that its normal human homolog is at dosage imbalance in Down syndrome (DS) . The human homolog of wv may also be involved in some forms of Parkinson's disease, as wv/wv mice display a deficit in the dopaminergic projection to the caudoputamen traced to degeneration of the tyrosine hydroxylase-positive cells of the substantia nigra beginning at postnatal day 7 (P7) (Schmidt et al. 1982). In addition to these central nervous system (CNS) anomalies, sperm in the wv/wv testis progress to the late spermatid stage, but mature sperm are never released (Harrison and RofflerTarlov 1993).The best-characterized aspect of the wv phenotype is the ataxia that results from a failure of granule cells (GC) to mature and migrate to form the cerebellar IGL. wv/wv individuals are first identifiable at the day of birth (P0) by an increased number of pyknotic nuclei in the external granule layer (EGL) of the cerebellum . By 8 days of age, both the lack of GC migration in mutant animals and differences in cerebellar size and organization among +/+, wv/+, and wv/wv mice are evident on histological analysis. Two distinct lines of investigation suggest that the wv defect is GC specific, but give rise to conflicting conclusions about whether the defect is extrinsic or intrinsic. Aggregate cultures of neurons (presumptive GC) cultured from the EGL of young normal mice extend neurites, whereas wv/wv aggregates do not. wv/wv cells will extend neurit...

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