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


Dr Fausto Cossu, Bone Marrow Transplant Unit,Ospedale, Microcitemico, Via Jenner s/n, 09121 Cagliari, Italy. E-mail: fcossu@mcweb.unica.it


Summary.  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+BNK’ 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.

X-linked Hoyeraal–Hreidarsson syndrome (XL-HHS; MIM 300240) is a severe multisystem disorder affecting male infants and characterized by growth retardation of prenatal onset, cerebellar hypoplasia, microcephaly, bone marrow (BM) failure and immunodeficiency. Recent demonstrations of mutations in the DKC1 gene in XL-HHS families have shown that it is a severe variant of X-linked dyskeratosis congenita (XL-DC; MIM 305000). These infants die prematurely before developing the typical DC mucocutaneous triad (leucoplakia, nail dystrophy and abnormal skin pigmentation) (Knight et al, 1999; Dokal, 2000). To date, only two XL-HHS families have been described which have missense mutations in the DKC1 gene (Thr49Met and Ser121Gly). It is noteworthy that the common recurrent mutation Ala353Val, which accounts for approximately 30% of all X-linked DC, was also found in a 4-year-old boy with overlapping features of HHS and DC (Yaghmai et al, 2000).

We report a novel DKC1 mutation in a newly diagnosed XL-HHS Sardinian infant, who showed the typical manifestations of this syndrome, including the recently recognized ‘T+BNK’ severe combined immunodeficiency (SCID) (Knight et al, 1999). He underwent sibling bone marrow transplantation (BMT), using a conditioning regimen chosen on the basis of HHS/DC phenotype, and at 1 year post transplant shows complete engraftment and correction of the disease haematological phenotype.

Case report

A 9-month-old Sardinian male infant was referred to the Ospedale Microcitemico in October 2000 with failure to thrive and delayed psychomotor development. His parents were unrelated. He was born at 36 weeks gestation with intrauterine growth retardation (birth weight 1·43 kg). Clinical examination at 9 months showed thin sparse scalp hair, microcephaly and tongue leucoplakia. Investigations demonstrated anaemia (Hb 7·5 g/dl, with 25% HbF) and thrombocytopenia (platelets < 20 × 109/l) with a hypocellular BM. The head nuclear magnetic resonance scan showed marked hypoplasia of cerebellar vermis and hemispheres. His clinical course was complicated by persistent regurgitation, diarrhoea, and recurrent bacterial and Candida infections. He went on to develop Pneumocystis carinii pneumonia, which was treated with Co-trimoxazole therapy and mechanical ventilation. Further investigations demonstrated agammaglobulinaemia and lymphopenia with absence of B lymphocytes and natural killer (NK) cells, but with a relatively normal numbers of T lymphocytes (Table I).

Table I.  Immunological data (blood).
3 months6 months12 months
  • *

    Supported by human IgG i.v. infusions.

  • cpm, counts per minute; PHA, phytohaemagglutinin; IL-2, interleukin 2; SEB, Staphylococcal enterotoxin B (superantigen); alloantigen, irradiated human pool mononuclear cells.

Ig (g/l)
 IgM< 0·00040·120·250·790·40–2·60
 IgA< 0·00060·100·160·240·10–1·60
Lymphocytes (× 109/l)2·011·241·721·983·60–8·30
T cells (× 109/l)
 CD31·98 (99%)0·84 (67%)1·15 (67%)1·23 (62%)2·30–6·45
 CD41·45 (72%)0·49 (40%)0·90 (52%)0·93 (47%)1·02–3·60
 CD80·52 (26%)0·30 (24%)0·22 (13%)0·27 (14%)0·57–2·23
B cells (× 109/l)
 CD190·004 (0%)0·14 (11%)0·29 (17%)0·31 (16%)0·50–1·50
NK cells (× 109/l)
 CD16/560·008 (0%)0·24 (19%)0·24 (14%)0·41 (22%)0·30–0·83
T-cell proliferation (cpm × 103)

Based on the features of intrauterine growth retardation, developmental delay, microcephaly, bone marrow failure, cerebellar hypoplasia and immunodeficiency, a diagnosis of XL-HHS was made. Single-strand conformation polymorphism screening followed by direct sequence analysis, as previously described (Knight et al, 2001), led to the identification of a novel DKC1 mutation in exon 3 where a T→C change at nucleotide 113 results in Ile38Thr substitution in dyskerin. This mutation, not reported in DC/HH patients before (Heiss et al, 2001), was not detected in 50 normal controls. Subsequent analysis of his mother showed her to be heterozygous for this mutation while his 4-year-old sister was normal.

In January 2001 (at Ospedale Microcitemico), the infant had a BMT with his human leucocyte antigen (HLA)-identical normal sister as the donor. In view of the previous experience of high toxicity in DC/HH patients undergoing BMT, the conditioning was chosen to avoid busulphan and radiotherapy. This consisted of fludarabine 40 mg/m2/d for 5 d (d −7 to −3), rabbit antithymocyte globulin 2·5 mg/kg/d for 2 d (d −4 to −3) and melphalan 40 mg/m2 (d −2). Graft-versus-host disease (GVHD) prophylaxis was with oral cyclosporine (1 mg/kg/d) from d −2 and methylprednisolone 0·5 mg/kg/d i.v. from d −1. He was also given low-molecular-weight heparin (100 IU/kg/d i.v) to prevent veno-occlusive disease as there is evidence for endothelial activation in DC patients.

The total number of BM nuclear cells infused was 6·6 × 108/kg. Neutrophil engraftment (> 0·5 × 109/l) occurred on d +11 and he had an unsupported platelet count of > 20 × 109/l from d +15. DNA analysis of peripheral blood at 0·5, 1, 3, 6, 9 and 12 months post BMT showed full (100%) donor haemopoiesis.

The post-transplant course was complicated by grade 2 acute GVHD of the skin. This responded to methylprednisolone (2 mg/kg/d). In addition, he received oral cyclosporine (4 mg/kg/d) and then 5 d (from d +92) of oral tacrolimus (0·5 mg/d) because of vomiting and diarrhoea which were initially thought to represent gut GVHD. The tacrolimus (and any calcineurin inhibitor) were stopped because of bilateral cortical blindness, which resolved completely after 6 weeks. Gastro-intestinal (GI) biopsies did not show GVHD; the GI symptoms, therefore, probably represented poor healing response to the stress of the conditioning.

In January 2002, 12 months post BMT, the blood count was normal (Hb 10·4 g/dl, white blood cell count 8·5 × 109/l, platelets 290 × 109/l) with adequate immune reconstitution (Table I). There had also been improvement in his psychomotor development. He had mucosal leucoplakia and developed nail dystrophy (first appeared from about d +150). His GI symptoms (oesophageal reflux, chronic diarrhoea) were slowly improving but he was still requiring parenteral nutrition.


This patient showed all the characteristic features of HHS as well as two out of the three features of the mucocutaneous triad of classical DC. He, therefore, represents one of the severest clinical phenotypes associated with mutant dyskerin. Dyskerin is a highly conserved 514-amino acid nucleolar protein. DKC1 is expressed in all human tissues and no null DKC1 mutations have been observed, suggesting they would be lethal. Dyskerin is associated with the H/ACA class of small nucleolar RNAs (snoRNAs) and is involved in the pseudouridylation of specific residues of ribosomal RNA. Dyskerin also associates with the RNA component (hTR) of telomerase, which is important in telomere maintenance (Collins, 2000). It has been recently shown that hTR is mutated in autosomal dominant DC (Vulliamy et al, 2001a). As dyskerin and hTR are both components of the telomerase complex and all subtypes of DC have short telomeres (Vulliamy et al, 2001b), it now appears that DC is principally a disease of defective telomere maintenance. Affected tissues are those that need constant renewal (including BM, skin, oral and gut epithelium, lung alveolar epithelium) consistent with a basic deficiency in stem cell activity due to defective telomerase activity.

Telomerase function is also important in development, differentiation and activation of human lymphocytes, and a variable number of immunological abnormalities can occur in DC patients (Dokal, 2000). The observation of impaired germinal centre reaction in mice with short telomeres (Herrera et al, 2000) provides further support for the importance of telomerase in lymphocyte function. Our patient had SCID characterized by the absence of B cells (with severe pan-hypogammaglobinaemia) and NK cells but relatively well-preserved T lymphocytes. This unusual ‘T+BNK’ SCID phenotype has been seen in other HHS patients and now appears to define a discrete subset of DC/HHS patients (Knight et al, 1999; Revy et al, 2000) (Table II).

Table II.  Phenotypes based on main lymphocyte subset counts in SCIDs and other PIDs.
T cellsB cellsNK cellsPrimary immunodeficiencies
  1. () absent or very low counts; (+) normal, elevated, or little reduced counts; SCID, severe combined immunodeficiency defined as a profound impairment of both cellular and humoral immunity causing death from opportunistic infections usually within the first year of life unless prevented by a timely successful haematopoietic stem-cell transplantation; PID, primary immunodeficiency; ADA, adenosine deaminase; RAG, recombinase activating gene; γc-chain, common cytokine receptor γ-chain; XSCID, X-linked SCID; JAK3, Janus kinase 3; IL-7Rα, interleukin 7 receptor α-chain; XLA, X-linked agammaglobulinaemia.

TBNKInfantile onset ADA deficiency
Reticular dysgenesis
TBNK+Complete RAG-1 or RAG-2 deficiency
RAG-independent V(D)J recombination defects
(ARTEMIS deficiency)
TB+NKγc-chain deficiency XSCID
JAK3 deficiency
CD45 deficiency
TB+NK+IL-7Rα deficiency
T+B+NK+T-cell signal transduction/calcium flux defects
T+BNK+Omenn syndrome
TBNK+ SCID with mother T-cell engraftment
T+BNKHoyeraal–Hreidarsson syndrome
TBNKSCID with mother T-cell engraftment
 Other PIDs
T+B+NKIsolated NK cell deficiency
T+BNK+Bruton's XLA
Autosomal recessive agammaglobulinaemia

Because of the severe immunodeficiency and BM failure, an early BMT was performed. He is the youngest patient to be transplanted for HHS. Previously, one BMT has been reported in a HHS patient and his course was characterized by severe complications (Knight et al, 1999). Furthermore BMT using conventional conditioning regimens in other DC patients has also been associated with high mortality, particularly related to pulmonary/vascular complications. In view of these reports together with the early genetic diagnosis in this patient, we chose a non-busulphan, non-total body irradiation-containing regimen, consisting of fludarabine, rabbit antithymocyte globulin and low-dose melphalan (40 mg/m2) (modified from Amrolia et al, 2000). Such a low-intensity conditioning regimen would now also appear to be desirable given the molecular defect in telomerase in all DC/HHS cells. All tissues are likely to experience more toxicity compared with normal cells and have delayed healing following BMT conditioning, with the greatest impact being on those tissues with a rapid turn over. In this regard, the GI symptoms seen in this patient are likely to have represented delayed healing of the gut tissue. It is also noteworthy that, mTR–/– mice (in which both alleles of the gene encoding telomerase RNA have been knocked out) have a reduced capacity to respond to stresses such as wound healing and haemopoietic ablation (Rudolph et al, 1999).

In summary, this low-intensity conditioning regimen was associated with prompt and sustained engraftment and low toxicity despite the severe phenotypic abnormalities present in our patient. At 1-year post BMT, he had full donor haemopoiesis and good immunological reconstitution. In view of the very young age at the time of BMT, together with the recently recognized plasticity of haemopoietic stem cells, it will be interesting to see whether there will be any favourable effect on non-haemopoietic tissues on a longer follow-up.