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Mutations in genes encoding proteins that are involved in mitochondrial heme synthesis, iron-sulfur cluster biogenesis, and mitochondrial protein synthesis have previously been implicated in the pathogenesis of the congenital sideroblastic anemias (CSAs). We recently described a syndromic form of CSA associated with B-cell immunodeficiency, periodic fevers, and developmental delay (SIFD). Here we demonstrate that SIFD is caused by biallelic mutations in TRNT1, the gene encoding the CCA-adding enzyme essential for maturation of both nuclear and mitochondrial transfer RNAs. Using budding yeast lacking the TRNT1homolog, CCA1,we confirm that the patient-associated TRNT1mutations result in partial loss of function of TRNT1 and lead to metabolic defects in both the mitochondria and cytosol, which can account for the phenotypic pleiotropy.

Five spontaneous, allelic mutations in the α-spectrin gene, Spna1, have been identified in mice (spherocytosis [sph], sph1J, sph2J, sph2BC, sphDem). All cause severe hemolytic anemia. Here, analysis of 3 new alleles reveals previously unknown consequences of red blood cell (RBC) spectrin deficiency. In sph3J, a missense mutation (H2012Y) in repeat 19 introduces a cryptic splice site resulting in premature termination of translation. In sphIhj, a premature stop codon occurs (Q1853Stop) in repeat 18. Both mutations result in markedly reduced RBC membrane spectrin content, decreased band 3, and absent β-adducin. Reevaluation of available, previously described sphalleles reveals band 3 and adducin deficiency as well. In sph4J, a missense mutation occurs in the C-terminal EF hand domain (C2384Y). Notably, an equally severe hemolytic anemia occurs despite minimally decreased membrane spectrin with normal band 3 levels and present, although reduced, β-adducin. The severity of anemia in sph4Jindicates that the highly conserved cysteine residue at the C-terminus of α-spectrin participates in interactions critical to membrane stability. The data reinforce the notion that a membrane bridge in addition to the classic protein 4.1-p55-glycophorin C linkage exists at the RBC junctional complex that involves interactions between spectrin, adducin, and band 3.

X-linked sideroblastic anemia with ataxia (XLSA/A) is a rare syndromic form of inherited sideroblastic anemia associated with spinocerebellar ataxia, and is due to mutations in the mitochondrial ATP-binding cassette transporter Abcb7. Here, we show that Abcb7 is essential for hematopoiesis and formally demonstrate that XLSA/A is due to partial loss of function mutations in Abcb7 that directly or indirectly inhibit heme biosynthesis.

We identified a family with gray platelet syndrome (GPS) segregating as a sex-linked trait. Affected males had a mild bleeding disorder, thrombocytopenia, and large agranular platelets characteristic of GPS, while obligate carrier females were asymptomatic but had dimorphic platelets on peripheral smear. Associated findings included mild erythrocyte abnormalities in affected males. Linkage analysis revealed a 63 cM region on the X chromosome between markers G10578and DXS6797, which segregated with the platelet phenotype and included the GATA1gene. Sequencing of GATA1revealed a G-to-A mutation at position 759 corresponding to amino acid change Arg216Gln. This mutation was previously described as a cause of X-linked thrombocytopenia with thalassemia (XLTT) but not of gray platelet syndrome. Our findings suggest that XLTT is within a spectrum of disorders constituting the gray platelet syndrome, and we propose that GATA1 is an upstream regulator of the genes required for platelet α-granule biogenesis.

Diamond Blackman Anemia (DBA) is a severe erythroid hypoplasia that usually presents during the first year of life. Most cases are due to heterozygous mutations in ribosomal protein genes including RPS19, RPL5and RPL11. Ribosomal stress-induced p53 activation is thought to play a major role in DBA, leading to apoptosis and/or cell cycle arrest in the erythroid lineage. This response is dependent on accumulation of RPL5 and RPL11 subunits that bind and sequester MDM2, the ubiquitin ligase that naturally degrades p53. Thus, it is difficult to understand how this pathway can operate in the presence RPL5or RPL11mutations. Here we show that Rpl11 haploinsufficiency in mice leads to severe anemia characteristic of DBA and, surprisingly, p53 activation in the bone marrow.

Abstract 629

Gray Platelet Syndrome (GPS) is characterized by a variable, mild bleeding diathesis, associated with thrombocytopenia and large, agranular, functionally abnormal platelets. While the disorder has been well-described both biochemically and pathologically, to our knowledge, no genetic mutation has been associated with the disease. Most cases are sporadic, with a few sibships and apparent autosomal kindreds reported. In our investigation, the proband is a healthy 1 year-old female whose father and uncle had recently been diagnosed with GPS. The child had a platelet count of 382 x103platelets/μl, comprised of a dimorphic population of normal and large, agranular forms. Her CBC and peripheral blood smear were otherwise unremarkable. We subsequently analyzed four generations of this family. In addition to the proband's father and uncle, several other members had known bleeding tendencies. The symptomatic individuals were invariably males, consistent with a sex-linked pattern of inheritance. Several symptomatic and asymptomatic family members were evaluated by CBC and blood smear. Large, agranular platelets were found in symptomatic males, while obligate carrier females exhibited both normal and abnormal forms. Using a set of fifteen microsatellite markers spanning the X chromosome, a common haplotype was identified in all affected men, their mothers, and their daughters. Aided by the published sequence of the X chromosome, we examined this region for candidate genes. Although the common haplotype between markers GATA144D04 and DXS6797 (Xp11.3-Xq22.3) contains hundreds of genes, only two, GATA1and WAS, have known associations with thrombocytopenia. PCR amplification and sequencing of a 3′ segment of the GATA-1 promoter and the five coding exons of the GATA-1 gene revealed an G759A missense mutation resulting in an Arg216Gln substitution in exon 4 (NCBI RefSeq: NM_002049) that segregated with the phenotype and was present in all obligate carrier females. This mutation has been previously associated with X-linked thrombocytopenia and beta-thalassemia (XLTT), a syndrome characterized by splenomegaly, thrombocytopenia, and imbalanced globin chain synthesis (Balduini et al, Thromb Haemost . 2004; 91:129). Comparison of the ultrastructural characteristics of the platelets in both disorders and a review of literature on both diseases suggests that XLTT, and the associated mutation in GATA1, could represent one genetic origin for GPS. As carrier females generally display a less severe phenotype than affected males, without thrombocytopenia and with subtle dimorphism on smears, it is possible that this mutation may account for some previously reported “sporadic” cases of GPS.