Cell and molecular biology

DC has many features in common with FA, in which cells display hypersensitivity to clastogenic agents such as MMC. Although the occasional DC patient may show some evidence of chro mosome breakage, in general there is no significant difference in chromosomal breakage between DC and normal lymphocytes with or without the use of bleomycin, DEB, MMC and y-irradiation. This observation enables DC patients to be distinguished from FA.

Primary DC skin fibroblasts are abnormal both in morphology and in growth rate. Furthermore, they show unbalanced chromosomal rearrangements (dicentrics, tricentrics, translocations) in the absence of any clastogenic agents. In addition, peripheral blood (PB) and BM metaphases from some patients show numerous unbalanced chromosomal rearrangements in the absence of any clastogenic agents. These studies provide evidence for a defect that predisposes DC cells to developing chromosomal rearrangements. DC, like FA, may thus be regarded as a chromosomal instability disorder but with a predisposition principally to chromosomal rearrangements rather than the gaps and breaks seen in FA.

Haemopoietic progenitor studies have shown reduced numbers of all progenitors compared with control subjects and there is usually a downward decline with time. The degree to which the progenitors are reduced can vary from patient to patient and they can be reduced even when the PB count is normal. The demonstration of abnormalities of growth and chromosomal rearrangements in fibroblasts suggests that the BM failure is likely to be a consequence of abnormalities in both haemo-poietic stem cells and stromal cells.

X-chromosome inactivation patterns (XCIPs) have been studied in PB cells of women from X-linked DC families by investigating a methylation-sensitive restriction enzyme site in the polymorphic human androgen receptor locus (HUMARA) at Xq11.2-Xq12. All carriers of X-linked DC showed complete skewing in XCIP. The presence of the extremely skewed pattern of X-inactivation in PB cells suggests that cells expressing the defective gene have a growth/survival disadvantage over those expressing the normal allele. Furthermore, a skewed XCIP provides important information about carrier status for use in the counselling of families at risk of DC. In addition, XCIPs data allow us to distinguish an inherited mutation from a de novo event in sporadic male DC cases, as well as autosomal from X-linked forms of the disease.

The majority of DC patients recruited on the DCR (Dyskeratosis Congenita Registry, Hammersmith Hospital, London) are male. This observation suggests that the X-linked recessive form of DC represents a major subset of cases. Initially, through linkage analysis in one large family with only affected males, it was possible to map the gene for the X-linked form to Xq28. The availability of polymorphic genetic markers from the Xq28 and additional X-linked families facilitated positional cloning of the gene (DKC1) that is mutated in X-linked DC. The identification of the DKC1 gene in 1998 made available a genetic test that can be used to confirm diagnosis in suspected cases and antenatal diagnosis in X-linked families. It also led to the demonstration that another rare syndrome, the Hoyeraal-

Hreidarsson (HH) syndrome, is due to mutations in the DKC1 gene. HH is a severe multisystem disorder characterized by severe growth failure, abnormalities of brain development (usually cerebellar hypoplasia), aplastic anaemia and immunodeficiency (T + B-NK- severe combined immunodeficiency). The recognition that HH is a severe variant of DC has further highlighted the considerable variability of the DC phenotype.

In addition to providing an accurate diagnostic test, this genetic advance has provided insights into the pathogenesis of DC. The DKC1 gene is expressed in all tissues of the body, indicating that it has a vital 'housekeeping function' in the human cell. This correlates well with the multisystem phenotype of DC. The DKC1 gene and its encoded protein, dyskerin, are highly conserved throughout evolution. Dyskerin is a nucleolar protein that is associated with the H/ACA class of small nucleolar RNAs (snoRNAs) and is involved in pseudo-uridylation of specific residues of ribosomal RNA (rRNA). This step is essential for ribosome biogenesis and therefore initially suggested that DC arises largely because of defective ribosome biogenesis.

Subsequent studies have shown that dyskerin also associates with the RNA component of telomerase (hTR), which also contains an H/ACA consensus sequence. Telomerase is an enzyme complex that is important in maintaining the telomeres of chromosomes. The precise composition of the telomerase complex is unknown, but two essential components, the RNA component (hTR) and the catalytic reverse transcriptase (hTERT), have been well characterized. Telomerase activity can be reconstituted in vitro using just the hTR and hTERT. In patients with X-linked DC, it was initially demonstrated that the level of hTR was reduced and that telomere lengths were much shorter than in age-matched normal control subjects. Subsequently, it was found that telomeres are also shorter in cells from patients with autosomal forms of DC. This therefore suggested that DC might principally be a disease of telomere maintenance rather than of ribosomal biogenesis. Further clarification came from linkage analysis in one large DC family, which showed that the gene for autosomal dominant DC is on chromosome 3q, in the same area where the gene for hTR had been previously mapped. This led to hTR mutation analysis in this and other DC families and the demonstration that autosomal dominant DC is due to mutations in the hTR gene.

Htr Telomerase

Figure 12.5 Putative associations between dyskerin and hTR (human telomerase RNA) in the telomerase complex. hTERT represents human telomerase reverse transcriptase. GAR1, NHP2 and NOPIO are ribo-nucleoproteins (RNPs) that are known to associate with dyskerin and the H/ACA class of small nucleolar RNAs (snoRNAs).

Figure 12.5 Putative associations between dyskerin and hTR (human telomerase RNA) in the telomerase complex. hTERT represents human telomerase reverse transcriptase. GAR1, NHP2 and NOPIO are ribo-nucleoproteins (RNPs) that are known to associate with dyskerin and the H/ACA class of small nucleolar RNAs (snoRNAs).

As the DKC1 -encoded protein dyskerin and hTR are both components of the telomerase complex (Figure 12.5), it now appears that DC arises principally from an abnormality in telomerase activity. Affected tissues are those that need constant renewal, consistent with a basic deficiency in stem cell activity due to defective telomerase activity. The demonstration of DKC1 and hTR mutations in DC families provides an accurate diagnostic test, including antenatal diagnosis, in a significant subset of cases (Table 12.6). For DC patients, this now also provides the basis for designing new treatments. For the wider community it provides the first direct genetic link between a human disease that is characterized by features of premature ageing (premature grey hair/hair loss, early dental loss, abnormalities of skin pigmentation, nail dystrophy, bone marrow failure,

Table 12.6 Dyskeratosis congenita genetic subtypes.

DC subtype

Approximate percentage of DC patients

Chromosome location

Gene product

Mutations identified

X-linked recessive





Autosomal dominant





Autosomal recessive*





*The genetic basis of this subtype is unknown and may represent more than one genetic locus. Data are based on the DCR registry.

*The genetic basis of this subtype is unknown and may represent more than one genetic locus. Data are based on the DCR registry.

increased predisposition to malignancy) and short telomeres. Therefore, unravelling the biology of this rare disease has important implications not only for patients with DC but also for the more common disorders, such as ageing, cancer and AA, that are also associated with abnormal telomeres. It is noteworthy that hTR mutations have been identified recently in patients with AA but who lacked diagnostic features of DC. Furthermore, AA patients with hTR mutations had significantly shorter telomeres than age-matched control subjects. These data indicate that in a subset of patients with AA the disorder is associated with a genetic lesion in the telomere maintenance pathway. They also highlight the diverse manifestations of DC: its severe variant form as the HH syndrome, its classical form and its mildest form as ' aplastic anaemia'. The similarities between DC and AA are given in Table 12.2.

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