The genomics of inherited bone marrow failure: from mechanism to the clinic

The inherited bone marrow failure syndromes (IBMFS) typically present with significant cytopenias in at least one haematopoietic cell lineage that may progress to pancytopenia, and are associated with increased risk of cancer. Although the clinical features of the IBMFS are often diagnostic, variable disease penetrance and expressivity may result in diagnostic dilemmas. The discovery of the genetic aetiology of the IBMFS has been greatly facilitated by next‐generation sequencing methods. This has advanced understanding of the underlying biology of the IBMFS and been essential in improving clinical management and genetic counselling for affected patients. Herein we review the clinical features, underlying biology, and new genomic discoveries in the IBMFS, including Fanconi anaemia, dyskeratosis congenita, Diamond Blackfan anaemia, Shwachman Diamond syndrome and some disorders of the myeloid and megakaryocytic lineages.     

Comparative analysis of Shwachman‐Diamond syndrome to other inherited bone marrow failure syndromes and genotype–phenotype correlation

Hashmi SK, Allen C, Klaassen R, Fernandez CV, Yanofsky R, Shereck E, Champagne J, Silva M, Lipton JH, Brossard J, Samson Y, Abish S, Steele M, Ali K, Dower N, Athale U, Jardine L, Hand JP, Beyene J, Dror Y. Comparative analysis of Shwachman‐Diamond syndrome to other inherited bone marrow failure syndromes and genotype–phenotype correlation. Our knowledge of the phenotypes of inherited bone marrow failure syndromes (IBMFSs) derives from case reports or case series in which only one IBMFS was studied. However, the substantial phenotypic overlap necessitates comparative analysis between the IBMFSs. Shwachman–Diamond syndrome (SDS) is an IBMFS that the appreciation of what comprises its clinical phenotype is still evolving. In this analysis we used data on 125 patients from the Canadian Inherited Marrow Failure Study (CIMFS), which is a prospective multicenter population‐based study. Thirty‐four cases of SDS patients were analyzed and compared to other patients with the four most common IBMFSs on the CIMFS: Diamond Blackfan anemia, Fanconi anemia (FA), Kostmann/severe congenital neutropenia and dyskeratosis congenita (DC). The diagnosis of SDS, FA and DC was often delayed relative to symptoms onset; indicating a major need for improving tools to establish a rapid diagnosis. We identified multiple phenotypic differences between SDS and other IBMFSs, including several novel differences. SBDS biallelic mutations were less frequent than in previous reports (81%). Importantly, compared to patients with biallelic mutations, patients with wild type SBDS had more severe hematological disease but milder pancreatic disease. In conclusion, comprehensive study of the IBMFSs can provide useful comparative data between the disorders. SBDS‐negative SDS patients may have more severe hematological failure and milder pancreatic disease.     

A role for heterochromatin protein 1γ at human telomeres

Human telomere function is mediated by shelterin, a six-subunit complex that is required for telomere replication, protection, and cohesion. TIN2, the central component of shelterin, has binding sites to three subunits: TRF1, TRF2, and TPP1. Here we identify a fourth partner, heterochromatin protein 1γ (HP1γ), that binds to a conserved canonical HP1-binding motif, PXVXL, in the C-terminal domain of TIN2. We show that HP1γ localizes to telomeres in S phase, where it is required to establish/maintain cohesion. We further demonstrate that the HP1-binding site in TIN2 is required for sister telomere cohesion and can impact telomere length maintenance by telomerase. Remarkably, the PTVML HP1-binding site is embedded in the recently identified cluster of mutations in TIN2 that gives rise to dyskeratosis congenita (DC), an inherited bone marrow failure syndrome caused by defects in telomere maintenance. We show that DC-associated mutations in TIN2 abrogate binding to HP1γ and that DC patient cells are defective in sister telomere cohesion. Our data indicate a novel requirement for HP1γ in the establishment/maintenance of cohesion at human telomeres and, furthermore, may provide insight into the mechanism of pathogenesis in TIN2-mediated DC.     

The H/ACA RNP assembly factor SHQ1 functions as an RNA mimic

SHQ1 is an essential assembly factor for H/ACA ribonucleoproteins (RNPs) required for ribosome biogenesis, pre-mRNA splicing, and telomere maintenance. SHQ1 binds dyskerin/NAP57, the catalytic subunit of human H/ACA RNPs, and this interaction is modulated by mutations causing X-linked dyskeratosis congenita. We report the crystal structure of the C-terminal domain of yeast SHQ1, Shq1p, and its complex with yeast dyskerin/NAP57, Cbf5p, lacking its catalytic domain. The C-terminal domain of Shq1p interacts with the RNA-binding domain of Cbf5p and, through structural mimicry, uses the RNA-protein-binding sites to achieve a specific protein-protein interface. We propose that Shq1p operates as a Cbf5p chaperone during RNP assembly by acting as an RNA placeholder, thereby preventing Cbf5p from nonspecific RNA binding before association with an H/ACA RNA and the other core RNP proteins.     

Reconstitution and structural analysis of the yeast box H/ACA RNA-guided pseudouridine synthase

Box H/ACA ribonucleoprotein particles (RNPs) mediate pseudouridine synthesis, ribosome formation, and telomere maintenance. The structure of eukaryotic H/ACA RNPs remains poorly understood. We reconstituted functional Saccharomyces cerevisiae H/ACA RNPs with recombinant proteins Cbf5, Nop10, Gar1, and Nhp2 and a two-hairpin H/ACA RNA; determined the crystal structure of a Cbf5, Nop10, and Gar1 ternary complex at 1.9 Å resolution; and analyzed the structure–function relationship of the yeast complex. Although eukaryotic H/ACA RNAs have a conserved two-hairpin structure, isolated single-hairpin RNAs are also active in guiding pseudouridylation. Nhp2, unlike its archaeal counterpart, is largely dispensable for the activity, reflecting a functional adaptation of eukaryotic H/ACA RNPs to the variable RNA structure that Nhp2 binds. The N-terminal extension of Cbf5, a hot spot for dyskeratosis congenita mutation, forms an extra structural layer on the PUA domain. Gar1 is distinguished from the assembly factor Naf1 by containing a C-terminal extension that controls substrate turnover and the Gar1–Naf1 exchange during H/ACA RNP maturation. Our results reveal significant novel features of eukaryotic H/ACA RNPs.     

Telomere length measurement can distinguish pathogenic from non-pathogenic variants in the shelterin component, TIN2

Dyskeratosis congenita (DC) is a heterogeneous bone marrow failure syndrome with seven disease-causing genes identified to date, six of which are linked to telomere maintenance. Mutations in one of these genes (TINF2), which encodes a component of the shelterin complex, are associated with particularly short telomeres. Among the 224 consecutive patients with different forms of bone marrow failure (46 with DC, 122 with aplastic anaemia and 57 with some features of DC), we have identified 16 new families with variants in exon 6 of the TINF2 gene, eight of which are novel. We observe that the phenotype associated with these mutations extends to a severe early presentation, not always classified as DC. In addition, we see that some of the variants identified are not associated with short telomeres and are also found in asymptomatic individuals. In the absence of any direct functional assay, the data indicates that the telomere length measurement can inform us as to which variants in TINF2 are pathogenic and which may be non-pathogenic.     

Acquired monosomy 7 myelodysplastic syndrome in a child with clinical features suggestive of dyskeratosis congenita and IMAGe association

We describe a case of acquired monosomy 7 myelodysplastic syndrome (MDS) in a boy with congenital adrenocortical insufficiency, genital anomalies, growth delay, skin hyperpigmentation, and chronic lung disease. Some of his clinical manifestations were suggestive of dyskeratosis congenita (DC), while other features resembled IMAGe association. DC has been linked to mutations in telomere maintenance genes. The genetic basis of IMAGe association is unknown, although mice harboring a mutation in a telomere maintenance gene, Tpp1, have adrenal hypoplasia congenita. We considered the possibility that this patient has a defect in telomere function resulting in features of both DC and IMAGe association. Pediatr Blood Cancer 2010; 54:154–157. © 2009 Wiley‐Liss, Inc.     

Hoyeraal-Hreidarsson syndrome caused by a germline mutation in the TEL patch of the telomere protein TPP1

Germline mutations in telomere biology genes cause dyskeratosis congenita (DC), an inherited bone marrow failure and cancer predisposition syndrome. DC is a clinically heterogeneous disorder diagnosed by the triad of dysplastic nails, abnormal skin pigmentation, and oral leukoplakia; Hoyeraal-Hreidarsson syndrome (HH), a clinically severe variant of DC, also includes cerebellar hypoplasia, immunodeficiency, and intrauterine growth retardation. Approximately 70% of DC cases are associated with a germline mutation in one of nine genes, the products of which are all involved in telomere biology. Using exome sequencing, we identified mutations in Adrenocortical Dysplasia Homolog (ACD) (encoding TPP1), a component of the telomeric shelterin complex, in one family affected by HH. The proband inherited a deletion from his father and a missense mutation from his mother, resulting in extremely short telomeres and a severe clinical phenotype. Characterization of the mutations revealed that the single-amino-acid deletion affecting the TEL patch surface of the TPP1 protein significantly compromises both telomerase recruitment and processivity, while the missense mutation in the TIN2-binding region of TPP1 is not as clearly deleterious to TPP1 function. Our results emphasize the critical roles of the TEL patch in proper stem cell function and demonstrate that TPP1 is the second shelterin component (in addition to TIN2) to be implicated in DC.