Germline NPM1 mutations lead to altered rRNA 2'-O-methylation and cause dyskeratosis congenita.

RNA modifications are emerging as key determinants of gene expression. However, compelling genetic demonstrations of their relevance to human disease are lacking. Here, we link ribosomal RNA 2'-O-methylation (2'-O-Me) to the etiology of dyskeratosis congenita. We identify nucleophosmin (NPM1) as an essential regulator of 2'-O-Me on rRNA by directly binding C/D box small nucleolar RNAs, thereby modulating translation. We demonstrate the importance of 2'-O-Me-regulated translation for cellular growth, differentiation and hematopoietic stem cell maintenance, and show that Npm1 inactivation in adult hematopoietic stem cells results in bone marrow failure. We identify NPM1 germline mutations in patients with dyskeratosis congenita presenting with bone marrow failure and demonstrate that they are deficient in small nucleolar RNA binding. Mice harboring a dyskeratosis congenita germline Npm1 mutation recapitulate both hematological and nonhematological features of dyskeratosis congenita. Thus, our findings indicate that impaired 2'-O-Me can be etiological to human disease.

Differences in disease severity but similar telomere lengths in genetic subgroups of patients with telomerase and shelterin mutations.

The bone marrow failure syndrome dyskeratosis congenita (DC) has been considered to be a disorder of telomere maintenance in which disease features arise due to accelerated shortening of telomeres. By screening core components of the telomerase and shelterin complexes in patients with DC and related bone marrow failure syndromes we have identified 24 novel mutations: 11 in the RNA component of telomerase (TERC), 8 in the reverse transcriptase component (TERT), 4 in dyskerin (DKC1) and 1 in TRF1-interacting nuclear factor 2 (TINF2). This has prompted us to review these genetic subtypes in terms of telomere length, telomerase activity and clinical presentation among 194 genetically characterised index cases recruited onto the registry in London. While those with DKC1 and TINF2 mutations present at a younger age and have more disease features than those with TERC or TERT mutations, there is no difference in telomere length between these groups. There is no difference in the age of onset and numbers of disease features seen in those with TERC and TERT mutations despite the fact that the latter show higher levels of telomerase activity in vitro. The incidence of aplastic anaemia is greater in patients with TERC or TINF2 mutations compared to patients with DKC1 mutations, and cancer incidence is highest in patients with TERC mutations. These data are the first to provide robust comparisons between different genetic subtypes of telomerase and shelterin mutations (the "telomereopathies") and clearly demonstrate that disease severity is not explained by telomere length alone.

Mutations in dyskeratosis congenita: their impact on telomere length and the diversity of clinical presentation.

The two genes mutated in the bone marrow failure syndrome dyskeratosis congenita (DC) both encode components of the telomerase complex responsible for maintaining the ends of chromosomes in stem cells and in the germ line. In reviewing the mutation profile that is found in DC, we describe 9 novel mutations in the DKC1 gene and 3 novel TERC mutations responsible for the X-linked and autosomal dominant forms of the disease, respectively, but find that two thirds of the families do not have mutations in either of these genes. In a significant subset of these uncharacterized families, the index case presents with severe disease previously defined as the Hoyeraal Hreidarsson (HH) syndrome. The diverse clinical phenotype seen in patients with X-linked DC is not explained by the different amino acid substitutions: Presentation of the recurrent A353V substitution ranges from classic DC to the severe HH variant. However, we do see that patients with HH have significantly shorter telomeres than those with a relatively mild presentation. In the new families described with TERC mutations, there is further evidence of disease anticipation associated with shorter telomeres in the younger generations. This study highlights the considerable genetic and phenotypic diversity of DC.

Mutations in the reverse transcriptase component of telomerase (TERT) in patients with bone marrow failure.

Human telomerase has two core components, the RNA molecule (TERC) that provides the template for telomere repeat elongation and a reverse transcriptase (TERT) that is responsible for the addition of telomere repeats at the ends of each chromosome. Mutations in TERC have been found in the autosomal-dominant form of the inherited bone marrow failure syndrome dyskeratosis congenita and in a subset of patients with aplastic anemia and myelodysplasia. These patients have short telomeres compared to age-matched controls. These observations suggest that uncharacterised cases of dyskeratosis congenita/aplastic anemia may have mutations in TERT or other molecules that associate with TERC in the telomerase complex. We have therefore screened the TERT gene for mutation by denaturing HPLC in 80 patients with inherited and acquired bone marrow failure (24 with dyskeratosis congenita, 36 with constitutional aplastic anemia, 13 with idiopathic aplastic anemia and 7 with other forms of bone marrow failure). 15 different TERT mutations have been identified. Of these, 5 are in flanking intron sequences, 6 are synonymous and 4 are non-synonymous (missense) substitutions in the coding sequence. These are the first natural mutations of TERT to be described and we highlight their possible pathogenic role in the development of bone marrow failure.

A novel DKC1 mutation, severe combined immunodeficiency (T+B-NK- SCID) and bone marrow transplantation in an infant with Hoyeraal-Hreidarsson syndrome.

X-linked Hoyeraal-Hreidarsson syndrome (XL-HHS) is the severe infantile variant of X-linked dyskeratosis congenita (XL-DC) and both are due to mutations in the DKC1 gene within Xq28. We report a novel missense mutation in DKC1 exon 3 (T113-->C, Ile38Thr) in a Sardinian infant with XL-HHS in whom the disease was characterized by 'T+B-NK-' severe combined immunodeficiency and bone marrow failure. He underwent sibling bone marrow transplantation using a conditioning regimen (fludarabine, rabbit antithymocyte globulin, low-dose melphalan) selected according to the HHS/DC phenotype. This was associated with low toxicity, prompt engraftment with adequate immune reconstitution and full donor haemopoiesis.

Expression of Plasmodium falciparum G6PD-6PGL in laboratory parasites and in patient isolates in G6PD-deficient and normal Nigerian children.

As the production of NADPH in the pentose phosphate pathway is the main antioxidant defence mechanism available to the Plasmodium falciparum, we have studied the expression of P. falciparum glucose 6-phosphate dehydrogenase-6-phosphogluconolactonase (PfG6PD-6PGL) in G6PD-deficient and normal erythrocyte host cells. Both erythrocytes infected in vitro with a laboratory isolate and erythrocytes from natural human infections were used. Total RNA was prepared from parasites collected from five G6PD-deficient and nine G6PD-normal children in Ibadan, Nigeria, selected after screening 189 rural schoolchildren and 68 clinical malaria patients, and was subjected to Northern blot analysis. The probe was a cDNA fragment of the G6PD domain of the PfG6PD-6PGL gene, with an internal control probe of P. falciparum 18S ribosomal RNA. Quantification was performed using a phosphoimager. Relative to internal control, the abundance of PfG6PD-6PGL mRNA (mean +/- standard deviation) was lower in parasites from G6PD-deficient children (0.29 +/- 0.27) than in G6PD-normal control subjects (0.74 +/- 0.26) (P = 0.014, Mann-Whitney U-test). Although confirmation in a larger study is required, our results suggest a lower relative abundance of PfG6PD-6PGL, and presumably antioxidant activity, in malaria parasites from G6PD-deficient hosts, thus extending the current knowledge of the mechanism of G6PD-deficiency related host protection.

Three major glucose-6-phosphate dehydrogenase-deficient polymorphic variants identified in Mazandaran state of Iran.

We report the first investigation of glucose- 6-phosphate dehydrogenase (G6PD) deficiency among the Mazandaranians in the north of Iran. We analysed the G6PD gene in 74 unrelated G6PD-deficient men with a history of favism. Molecular analysis revealed three major different polymorphic variants: G6PD Mediterranean 66.2% (49 out of 74), G6PD Chatham 27% (20 out of 74), G6PD Cosenza 6.75% (5 out of 74). These findings indicated a higher prevalence of G6PD Chatham in this Iranian population than anywhere else in the world. In addition, the distribution of these G6PD variants is more similar to that found in an Italian population than in other Middle Eastern countries.

Combined glucose-6-phosphate dehydrogenase and glucosephosphate isomerase deficiency can alter clinical outcome.

Glucosephosphate isomerase (GPI) deficiency in humans is an autosomal recessive disorder, which results in nonspherocytic hemolytic anemia of variable clinical expression. A 4-year-old female with severe congenital hemolytic anemia had low red cell GPI activity of 15.5 IU/g Hb (50% of normal mean) indicating GPI deficiency. Subsequent DNA sequence analysis revealed a novel homozygous 921C to G mutation in the GPI gene sequence, predicting a Phe307 to Leu replacement. Strikingly, the red cell GPI activity in this patient was higher than that found in a second patient expressing the same GPI variant, with a more severe clinical phenotype. We propose that the hemolysis in the first patient may be modified by an accompanying deficiency of glucose-6-phosphate dehydrogenase (G6PD). The proband's red cell G6PD activity was reduced at 4.5 IU/g Hb (50% of normal mean) and molecular studies revealed heterozygosity for the G6PD Viangchan mutation and a skewed pattern of X-chromosome inactivation, producing almost exclusive expression of the mutated allele. The G6PD Viangchan variant is characterised by severe enzyme deficiency, but not chronic hemolysis. This study suggests that the metabolic consequences of a combined deficiency of GPI and G6PD might be responsible for a different clinical outcome than predicted for either defect in isolation.