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Providing essential information needed in clinical practice for the diagnosis and management of patients with blood disorders, this handbook covers haematological investigations and their interpretation, and commonly used protocols.

Telomere biology disorders are complex clinical conditions that arise due to mutations in genes required for telomere maintenance. Telomere length has been utilised as part of the diagnostic work-up of patients with these diseases; here, we have tested the utility of high-throughput STELA (HT-STELA) for this purpose. HT-STELA was applied to a cohort of unaffected individuals (n = 171) and a retrospective cohort of mutation carriers (n = 172). HT-STELA displayed a low measurement error with inter- and intra-assay coefficient of variance of 2.3% and 1.8%, respectively. Whilst telomere length in unaffected individuals declined as a function of age, telomere length in mutation carriers appeared to increase due to a preponderance of shorter telomeres detected in younger individuals (< 20 years of age). These individuals were more severely affected, and age-adjusted telomere length differentials could be used to stratify the cohort for overall survival (Hazard Ratio = 5.6 (1.5–20.5); p < 0.0001). Telomere lengths of asymptomatic mutation carriers were shorter than controls (p < 0.0001), but longer than symptomatic mutation carriers (p < 0.0001) and telomere length heterogeneity was dependent on the diagnosis and mutational status. Our data show that the ability of HT-STELA to detect short telomere lengths, that are not readily detected with other methods, means it can provide powerful diagnostic discrimination and prognostic information. The rapid format, with a low measurement error, demonstrates that HT-STELA is a new high-quality laboratory test for the clinical diagnosis of an underlying telomeropathy.

Dyskeratosis congenita is a premature aging syndrome characterized by muco-cutaneous features and a range of other abnormalities, including early greying, dental loss, osteoporosis, and malignancy. Dyskeratosis congenita cells age prematurely and have very short telomeres. Patients have mutations in genes that encode components of the telomerase complex (dyskerin, TERC, TERT, and NOP10), important in the maintenance of telomeres. Many dyskeratosis congenita patients remain uncharacterized. Here, we describe the analysis of two other proteins, NHP2 and GAR1, that together with dyskerin and NOP10 are key components of telomerase and small nucleolar ribonucleoprotein (snoRNP) complexes. We have identified previously uncharacterized NHP2 mutations that can cause autosomal recessive dyskeratosis congenita but have not found any GAR1 mutations. Patients with NHP2 mutations, in common with patients bearing dyskerin and NOP10 mutations had short telomeres and low TERC levels. SiRNA-mediated knockdown of NHP2 in human cells led to low TERC levels, but this reduction was not observed after GAR1 knockdown. These findings suggest that, in human cells, GAR1 has a different impact on the accumulation of TERC compared with dyskerin, NOP10, and NHP2. Most of the mutations so far identified in patients with classical dyskeratosis congenita impact either directly or indirectly on the stability of RNAs. In keeping with this effect, patients with dyskerin, NOP10, and now NHP2 mutations have all been shown to have low levels of telomerase RNA in their peripheral blood, providing direct evidence of their role in telomere maintenance in humans.

Dyskeratosis congenita (DC) is a rare inherited bone marrow failure syndrome, caused by genetic mutations that principally affect telomere biology. Approximately 35% of cases remain uncharacterised at the genetic level. To explore the genetic landscape, we conducted genetic studies on a large collection of clinically diagnosed cases of DC as well as cases exhibiting features resembling DC, referred to as ‘DC-like’ (DCL). This led us to identify several novel pathogenic variants within known genetic loci and in the novel X-linked gene, POLA1 . In addition, we have also identified several novel variants in POT1 and ZCCHC8 in multiple cases from different families expanding the allelic series of DC and DCL phenotypes. Functional characterisation of novel POLA1 and POT1 variants, revealed pathogenic effects on protein-protein interactions with primase, CTC1-STN1-TEN1 (CST) and shelterin subunit complexes, that are critical for telomere maintenance. ZCCHC8 variants demonstrated ZCCHC8 deficiency and signs of pervasive transcription, triggering inflammation in patients’ blood. In conclusion, our studies expand the current genetic architecture and broaden our understanding of disease mechanisms underlying DC and DCL disorders. Synopsis The evolving genetic landscape of inherited bone marrow failure syndrome dyskeratosis congenita reveals new pathogenic variants that broadens our understanding of current genetic and molecular mechanisms underlying this disorder. Several novel pathogenic variants within known susceptibility loci, as well as in the novel X-linked locus POLA1, are part of the evolving DC and DCL genetic landscape. Telomere maintenance is impacted by novel variants in POLA1 and POT1, while pervasive transcription and inflammation are caused by ZCCHC8 variants. Current knowledge on disease mechanisms beyond the regulation of long non-coding RNA TERC is extended by the clinical, genetic, and molecular similarity between DC and DCL cases. The evolving genetic landscape of inherited bone marrow failure syndrome dyskeratosis congenita reveals new pathogenic variants that broadens our understanding of current genetic and molecular mechanisms underlying this disorder.

Dyskeratosis congenita is a progressive bone-marrow failure syndrome that is characterized by abnormal skin pigmentation, leukoplakia and nail dystrophy 1 , 2 . X-linked, autosomal recessive and autosomal dominant inheritance have been found in different pedigrees. The X-linked form of the disease is due to mutations in the gene DKC1 in band 2, sub-band 8 of the long arm of the X chromosome (ref. 3 ). The affected protein, dyskerin, is a nucleolar protein that is found associated with the H/ACA class of small nucleolar RNAs and is involved in pseudo-uridylation of specific residues of ribosomal RNA 4 . Dyskerin is also associated with telomerase RNA (hTR) 5 , which contains a H/ACA consensus sequence 6 , 7 . Here we map the gene responsible for dyskeratosis congenita in a large pedigree with autosomal dominant inheritance. Affected members of this family have an 821-base-pair deletion on chromosome 3q that removes the 3′ 74 bases of hTR. Mutations in hTR were found in two other families with autosomal dominant dyskeratosis congenita.

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.

Fanconi anemia is a genetic disease characterized by genomic instability and cancer predisposition 1 . Nine genes involved in Fanconi anemia have been identified; their products participate in a DNA damage–response network involving BRCA1 and BRCA2 (refs. 2 , 3 ). We previously purified a Fanconi anemia core complex containing the FANCL ubiquitin ligase and six other Fanconi anemia–associated proteins 4 , 5 , 6 . Each protein in this complex is essential for monoubiquitination of FANCD2, a key reaction in the Fanconi anemia DNA damage–response pathway 2 , 7 . Here we show that another component of this complex, FAAP250, is mutant in individuals with Fanconi anemia of a new complementation group (FA-M). FAAP250 or FANCM has sequence similarity to known DNA-repair proteins, including archaeal Hef, yeast MPH1 and human ERCC4 or XPF. FANCM can dissociate DNA triplex, possibly owing to its ability to translocate on duplex DNA. FANCM is essential for monoubiquitination of FANCD2 and becomes hyperphosphorylated in response to DNA damage. Our data suggest an evolutionary link between Fanconi anemia–associated proteins and DNA repair; FANCM may act as an engine that translocates the Fanconi anemia core complex along DNA.

The bone marrow failure syndromes are a diverse group of rare genetic conditions. Mutations in telomere maintenance genes cause a large proportion of cases of dyskeratosis congenita. Our aim was to determine whether TA65, marketed as a telomerase activator, can correct this defect in vitro. We also investigated danazol, which improves peripheral blood counts in dyskeratosis congenita and Fanconi's anaemia by an unknown mechanism. We isolated T lymphocytes from the peripheral blood of 13 patients with bone marrow failure (seven dyskeratosis congenita, two Fanconi's anaemia, four uncharacterised) and ten age-matched controls. All 23 T lymphocyte cultures were incubated with TA65 (0·5 μg/mL and 0·05 μg/mL). Lymphocytes from nine patients and five controls were also incubated with danazol (dose range 0·048 ng/mL to 3 μg/mL). Cell proliferation was measured by neutral red assay (days 6 and 8) and nucleocounter (day 8). Telomerase activity of cells from six patients with dyskeratosis congenita was measured by TRAP (telomeric repeat amplification protocol) assay on day 8. We observed increased proliferation of T lymphocytes after treatment with TA65 0·5μg/mL and danazol 6 ng/mL (starting cell count 1 × 106, mean final count 7·64 × 106 with TA65 and 7·66 × 106 with danazol vs 7·31 × 106 with dimethyl sulfoxide [DMSO] only, as the control condition). Dyskeratosis congenita cells showed the most consistent results with TA65 treatment, with increased growth of five out of six cultures and overall relative proliferation of 1·033 (95% CI 1·001–1·064). Danazol improved cell growth most markedly in patients with Fanconi's anaemia (1·4 times increased proliferation over DMSO control). There was 1·23 times increased telomerase activity of T lymphocytes from patients with dyskeratosis congenita treated with TA65 0·5 μg/mL compared with DMSO controls. Treatment with danazol 1·2 ng/mL resulted in 1·45 times increased telomerase activity. This is the first time, to our knowledge, that these drugs have been studied in vitro in patients with bone marrow failure. Our results show a trend to increased proliferation of T lymphocytes from patients with different bone marrow failure syndromes treated with TA65 and danazol. In addition, our data show increased telomerase activity in dyskeratosis congenita cells treated with these drugs. Our study is limited by small patient numbers from this rare heterogeneous bone marrow failure group. However, these preliminary results provide an exciting basis for further studies to help establish the clinical utility of these drugs. Barts Charity (Academic Clinical Fellowship Support Grant).