CLINICAL AND GENETIC FEATURES OF DGA

DGA has now been redefined as a developmental disorder affecting structures which originate embryologically from the third and fourth pharyngeal pouches. There is considerable variability in the defects and in the spectrum of associated anomalies.8 ,9 The diversity of clinical features (partial versus complete forms) makes precise diagnostic criteria difficult to define.10 About 90% of patients with DGA present hemizygosity for a region of chromosome 22q11 which is commonly deleted.5 ,8 ,9 The acronym CATCH22 syndrome (cardiac defects, abnormal facies, thymic hypoplasia, cleft palate, hypocalcaemia) has been suggested to describe this clinical spectrum of conditions, now very broad, associated with 22q11 deletions.8-10

Although the clinical and pathological studies seemed clearly to indicate that our patient fulfils all the criteria for a diagnosis of DGA,9 we sought additional evidence at a genetic level. Cytogenetic studies showed a normal 46XX karyotype with no deletions or translocations. No microdeletions were found at chromosome 22q11.2 by using D22S75 and Cos40 probes in FISH studies. In addition, the proband's parents did not show the 22q11 microdelection using M51 and sc4.1 cosmid probes (analysis kindly performed by P A In't Veld, Rotterdam). Furthermore, polymorphic markers for chromosome 22 did not reveal hemizygosity at this level (data not shown). The 10p deletion syndrome presented in cases with partial DGA exhibits specific phenotype characteristics, particularly sensorineural hearing loss.7 ,11 However, hearing was apparently normal and no 10p deletions were found in our patient.

Although a molecular defect responsible for this anomaly was not detected it is possible that the presence of a chimerism (see below) could render the interpretation of studies with highly polymorphic markers more difficult. Maternal origin of the defect was strongly suspected because of the family history: multiple spontaneous abortions and neonatal deaths favour the hypothesis of a cryptic molecular defect and together with the other mildly affected family members (micrognathia) point to a dominantly inherited problem which was undetected.

IMMUNOLOGICAL FOLLOW UP STUDIES

Analysis of peripheral blood lymphocytes on admission, showed CD3⁺, CD4⁺, and CD8⁺ subsets to be within normal ranges. Subsequently, the T cell numbers (CD3⁺, TCRαβ⁺) gradually decreased, mainly as a result of a slow but progressive loss of CD4⁺lymphocytes. Despite the low number of T cells, almost all these cells (>80%) were CD45R/RO⁺ (normal range 29–44%). T cells also showed an “activated” phenotype (CD3⁺CD25⁺, 40% and CD3⁺HLA-DR⁺, 70%) (age matched normal ranges are 3–7% and 6–9%, respectively). Functional analysis revealed low T cell proliferative responses triggered by PHA as well as ConA (not shown). Proliferation to CD3/TCR or to PMA was also depressed with normal responses to PWM throughout the patient's life (not shown). Using a panel of mAb against TCR V region gene encoded epitopes, we showed that T cells displayed a greatly reduced TCR diversity, characterised by a significantly diminished proportion of the TCR Vβ8⁺ subset and an important expansion of the TCR Vβ5.2 and Vβ5.3 subsets (fig 3).

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Figure 3

TCR diversity analysis of patient's T cells. Patient's peripheral mononuclear cells were analysed by flow cytometry using the indicated anti-TCR V monoclonal antibodies. Results are expressed as % of TCR expressing cells.

In contrast, a progressive increase in CD19⁺ cells was evident. The last phenotypic analysis performed when the IgM monoclonal paraprotein was detected, showed a high proportion of B cells mainly expresssing surface IgM (CD19⁺, 63% and sIgM⁺, 69.8%; normal range is 7–17%). The patient was routinely monitored for immunoglobulin concentrations. During the first six months of her life, serum IgG and IgM concentrations were within normal limits for her age. However, IgE was notably raised from birth and remained high for approximately one and a half years. Coinciding with the falling IgE concentration, IgG increased progressively and concentrations reached 2.5 g/l. A monoclonal IgG λ chain was detected on electrophoretic analysis by immunofixation. IgA concentrations were within normal range—or just below normal—on all occasions. IgM concentrations were also normal or slightly above normal, except shortly before death when a second monoclonal gammapathy (IgM λ) appeared. The significance of this finding in our patient lies in the isotype change in the monoclonal immunoglobulin, from IgG to IgM; such class switching is extremely rare. Next, we evaluated the IgH gene rearrangement by a seminested PCR method but were unable to detect clonal lymphoid proliferation of B cells shortly after the immunofixation detection of the IgG monoclonal band. However, PCR analysis of IgH rearrangements showed one band clearly detectable in the gel in the 80–120 bp range which allowed us to classify it as monoclonal. This coincided with the appearance of the IgM monoclonal band and the existence of a high proportion of sIgM⁺ B cells (fig 4). We did not study whether maternal cells were functional in vitro, but proliferative responses to mitogens in this patient were probably decreased as a result of the low number of T lymphocytes.

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Figure 4

Clonal heavy chain gene rearrangements (lanes C1⁺ and C2⁺ are DNA extracted from the Namalwa B cell line and DNA from the erythroleukaemia K562 cell line, respectively. The P_LN lane is postmortem extracted DNA from the DGA patient's lymph nodes while P_PB1–3 represent DNA extracted from peripheral blood at three different points during the clinical course. NHL₁ and NHL₂ indicate DNA from two unrelated patients with non-Hodgkin's B cell lymphomas. C⁻ represents a negative control with no DNA added.

Antibody production was tested in vivo by immunisation with both pneumococcal polysaccharide vaccine and tetanus toxoid. No response was obtained against these antigens. Similarly, specific production of antibodies against viral antigens was also negative. Although routine serological studies did not detect antibodies to EBV, CMV, and HIV antigens, a postmortem search for DNA sequences in different tissues revealed the presence of EBV DNA sequences in spleen and lymph nodes as well as in a sample of frozen PBMC (not shown) but not in several other tissues tested (jejunal tissue, lung, skin). CMV and HIV were negative by PCR studies. This has been recently reported in two patients with DGA who died of an associated B cell lymphoma.12 ,13 In both cases malignant cells expressed EBV. The high number of oligoclonal B cells observed in our patient could be related to an EBV driven expansion of these cells. However, we could not show histopathological evidence of malignancy on postmortem examination.

DEMONSTRATION OF MATERNO-FETAL CHIMERISM

Despite the patient's clinical and pathological findings indicating DGA, many other abnormalities seen in our patient, such as jejunal villous atrophy, exfoliative erythroderma, Evans' syndrome, monoclonal gammapathy, B cell proliferation, and antinuclear antibodies could be better explained by a chronic semiallogeneic interaction between maternal lymphocytes and recipient cells. In addition, the immunological alterations found (high IgE, restricted heterogeneity of TCR diversity, low proliferative response of PBMC to mitogens) could be also explained by GVHD. This could be caused by transplacental passage of maternal lymphocytes as shown by the presence of maternal DNA in the patient's tissues by HLA typing of the family (table 1). These results clearly indicated a maternally derived cell engraftment in the patient.

OTHER DIAGNOSIS RELATED CONSIDERATIONS

Skin conditions, lymphadenopathy, hepatosplenomegaly, failure to thrive, chronic diarrhoea, recurrent infections, eosinophilia, increased IgE, and activated T cells with low proliferation are all features seen both in Omenn's syndrome and materno-fetal GVHD.14 Some authors have reported clinicopathological findings resembling those observed in GVHD which argue in favour of the hypothesis that a lymphocytic chimerism caused by intrauterine transfusion is responsible for Omenn's syndrome,15 but this has not been shown by other authors.16 ,17 There are reports of children with features of DGA with or without 22q11 deletion18 ,19 who developed Omenn's syndrome but in whom no maternal cells were present and patients with eczematous skin lesions occurring simultaneously with the appearance of a restricted Vβ TCR and without materno-fetal chimerism.20 ,21 Thymic defects, including those of DGA, may have a role in the mechanism contributing to the restricted T cell repertoire seen in Omenn's syndrome.22 Consequently, we also considered the possibility of Omenn's syndrome because of the clinical features (diarrhoea, failure to thrive, several episodes of generalised erythroderma, eosinophilia, liver, spleen and lymph node enlargement, together with repeated infections)15-17 23-25 and certain immunological features (activated T cells with low proliferative response and restricted heterogeneity of TCR diversity) present in our patient. However, to our knowledge, Omenn's syndrome could be ruled out in this case by because: (1)RAG1 and RAG2mutations were not detected in our patient (not shown); (2) increased percentages of B cells were present throughout the life of our patient; and (3) as stated, the presence of maternal cells in the patient was unequivocally shown, a fact that could explain the Omenn's syndrome like features.

CONCLUDING REMARKS

DGA is still an enigma.26 This case report is the first, to our knowledge, which presents the simultaneous occurrence of severe GVHD, including Evans' syndrome, two monoclonal gammopathies of undetermined significance,27 ,28 and other immunological dysfunctions in DGA. The early ocurrence of GVHD probably resulted in chronic allogeneic stimulation. This, together with the activation of residual T cell clones and lack of physiological programmed cell death in the thymus are linked processes which could trigger autoimmune manifestations (Evans' syndrome, ANA). The immunoregulatory imbalance characteristic of DGA together with the evidence of transplacental passage of maternal T cells could explain this varied clinical picture. In addition, recurrent infections including EBV could have contributed to her grave clinical evolution. This girl could be included in the small group of patients in which neither FISH nor PCR analysis of polymorphic markers detect chromosome deletions. This does not rule out the existence of undetected smaller deletions or point mutations within DGA critical region genes.