Main

The group of congenital heart diseases characterized by conotruncal abnormalities can fall into any of a number of related syndromes, all of which are linked to a deletion in chromosome 22.1 This group is typified by the DiGeorge syndrome and is usually associated with a wide spectrum of noncardiological anomalies. Although the chromosomal deletion has been known for some time, the identification of a single gene most likely to cause the diverse features of this phenotype has been difficult. However, recent advances in a distant, seemingly unrelated field - that of intracellular protein turnover - have led to a unified theory of the pathogenesis of this confusing group of disparate syndromes.

With this review, we will outline the history of the development of the various parallel syndromes that have the chromosome 22 deletion in common and will include a brief overview of the embryogenesis of the relevant congenital heart and associated anomalies. We will consider the role of the ubiquitin-proteasome pathway in their development and show how the deletion of a single gene may explain the wide array of abnormalities that are typical of these syndromes.

HISTORICAL BACKGROUND

The association between congenital thymic hypoplasia and hypocalcemia was first described by Lobdell in 1959 (Table 1).2 In 1968, DiGeorge included T-cell dysfunction. Only later3 were the typical cardiac and facial anomalies added, which are now considered to be the hallmarks of the well-known DiGeorge syndrome (DGS).

Table 1 Historical overview of the syndromes associated with the 22q11.2 deletion
Full size table

Kinouchi et al.4,5 soon afterward outlined the conotruncal anomaly face syndrome (also known as Takao syndrome), associating a typical facial appearance, velopharyngeal insufficiency, and cardiac abnormalities with learning disabilities. They saw this “new” syndrome as a forme fruste, or transitional form of DGS, and speculated that the two syndromes formed part of a clinical spectrum.6 They also pointed out the clinical overlap with the velocardiofacial syndrome (see below).

Almost simultaneously, Shprintzen et al.7 described children with cardiac, velopharyngeal, and facial abnormalities and dubbed this association the velocardiofacial syndrome (VCFS). The features of this syndrome have expanded over the past 20 years8,9 and now comprise an overwhelming array of 185 features and associations grouped into 20 categories.10 VCFS has an incidence of at least 1:4000, although Shprintzen estimates the birth incidence to be as high as 1:1800, making VCFS the second most common cause of congenital heart disease (after Down syndrome).

More recently, Cayler syndrome, first described in 1969,11 has been shown to be associated with the 22q11.2 deletion12 and its phenotypic spectrum has subsequently been enlarged to include multiple extracardiac anomalies.13

It seemed, therefore, that children with a wide array of congenital abnormalities in association with a characteristic spectrum of cardiac anomalies could be classified into any of a number of phenotypically similar syndromes and that the boundaries between these were being increasingly blurred.14 In 1989, Burn noted the similarity between these syndromes and suggested a synthesis. The common denominator between these phenotypes was first hinted at by De la Chapelle,15 who noted a 20:22 chromosomal translocation in a patient with features suggestive of DGS, and later confirmed by Kelley et al.16 as a deletion of the long arm of chromosome 22. Wilson et al.17 then summarized the salient features, using the acronym “CATCH-22” for:C ardiac disease, A bnormal faces, T hymic hypoplasia, C left palate, H ypocalcemia, associated with a deletion in chromosome-22. This acronym rapidly became common parlance, but Burn18 has subsequently appealed for discontinuing its use, since the vernacular term “CATCH-22”, taken from the title of Heller’s book,19 implies a “no-win situation” and is consequently a pejorative medical epithet.

THE DELETION

This hemizygous deletion (del 22q11.2) occurs in up to 89% of patients with the phenotypic features typical of DGS and may make up to 15% of all congenital heart diseases.20 Prevalence in the general population is approximately 1:4000, as shown by a large study of approximately 325,000 children in Belgium21 and correlates well with the incidence of VCFS.10 The deletion was initially underreported, since older cytogenetic studies were unable to detect the microdeletions in the 22q11.2 region which are now found by fluorescence in situ hybridization (FISH) probes.

Generally, the deletion is large (approximately 3-Mb22) and usually arises spontaneously, due to instability conferred by low copy repeats known to be situated at the deletion breakpoints,23 and within the 3-Mb deletion.24 The low copy repeats contain 200-kbp sequence duplications, which enable deletion (and duplication) of this region during homologous recombination events.

The deletion contains numerous genes, but linkage analysis has enabled the definition of a smaller region within this deletion as the so-called “critical region,” containing approximately 25 to 30 candidate genes. This has been termed the DiGeorge critical region (DGCR). Halford et al.25 have examined 114 deletions within this region and have been able to determine the smallest region of deletion overlap (SRO) to be 300 to 600 kbp.

CARDIAC EMBRYOGENESIS: CONTEXT AND CONTROL

Cardiac structures develop very early during embryogenesis and are derived from the cardiogenic cord in the embryonic mesoderm. The outflow structures of the heart (the conotruncus) are derived from branchial arches IV (aorta) and VI (pulmonary artery). Malalignment of these structures and incomplete septation give rise to the diverse congenital heart diseases associated with DGS. Lesions seen include arch abnormalities (interrupted aortic arch, aortic coarctation), conotruncal abnormalities (truncus arteriosus, pulmonary artery atresia), and malalignment of the interventricular septum with the conotruncus (VSD, ASD, tetralogy of Fallot, DORV). Abnormalities of the valvular structures or myocardium are unusual.

Much of the early organogenesis of the heart seems to be under control of neuroectodermal cells, derived from the neural crest, which migrate into the branchial arches very early during embryogenesis.26 Ablation of the cardiac neural crest in chicken embryos has been shown to lead to failure of outflow septation (truncus arteriosus or pulmonary atresia) and malalignment syndromes (TOF and DORV).27

The branchial arches are also the embryological origin of the anterior facial structures, and the thyroid, thymus, and parathyroids, and their organogenesis is equally under the control of neural crest cells. Deletion or abnormalities in development of these structures are the phenotypic hallmark of the DGS group.

How can the full phenotypic spectrum of all these syndromes with the myriad abnormalities of all these structures be explained in terms of the deletion of a single gene in the DGCR? The explanation may lie in the elegant control of apoptosis during embryogenesis by the ubiquitin-proteasome pathway.

UBIQUITINATION

Ubiquitination is a highly evolved, finely regulated process of controlled intracellular protein degradation.28,29 The ubiquitination pathway is present in all eukaryocytic cells and is the main process by which cells regulate intracellular protein turnover, allowing exact control of protein half-life and activity. The process also plays a role in intracellular housekeeping: damaged or redundant proteins are lysed to their peptide constituents by the pathway.

There are three major parts to the system:

  • 1. The ubiquitin molecule: a 76 amino acid protein which binds covalently to the target protein, conferring the “kiss of death” to that protein.

  • 2. The S26 proteasome: a large multienzyme complex which effects ATP-dependent proteolysis on ubiquitin-bound proteins.

  • 3. A complex array of controlling enzymes.

Ubiquitin was first described by Schlesinger et al.30 as a protein present in all eukaryocytic cells and soon after, its importance as the trigger for proteolysis was determined.31 The pathway was further elucidated in the past two decades, and now its role in such diverse processes as DNA repair, stress response, transmembrane transport, apoptosis,32 as well as cell differentiation and embryogenesis is better understood.33

Levels of the S26 proteasome are particularly high in areas and at times of rapid cell turnover, such as cancerous, lymphoblastic, and fetal cells.34 Wunsch and Haas35 have shown high concentrations of ubiquitin-protein conjugates in neuroectodermal cells, specifically neural crest cells and embryonic cardiac tissue, consistent with a critical role in the organogenesis of the branchial arches and heart.

It follows that any perturbation in this finely balanced system may have profound and widely divergent effects on the morphogenesis of the organs and structures that have their origin in the branchial arches or are under developmental control of the neuroectodermal cells. This has recently been shown. By using a screen for a transcription factor (dHAND) involved in neural crest development, Yamagishi et al.36 were able to identify a gene which is involved in ubiquitination, and which maps to 22q11.2 in the DGCR. This gene is called UFD1L (ubiquitin-fusion-degradation-1-like).

THE UFD1L GENE

The UFD1L gene is one of the 25 or so genes located in the DiGeorge critical region. This region is most consistently deleted in patients with DGS (88%) or VCFS (76%).37 Several genes in this region have been proposed in the past as candidate genes for the pathogenesis of the protean developmental abnormalities of the broader DGS phenotype, but none until now has fitted the description as well as the UFD1L gene.

In 1997, Pizzuti et al.38 highlighted this gene as a strong candidate for the deletion responsible for the DGS phenotype, by showing that the gene encodes a protein involved in the ubiquitin–proteasome pathway. Furthermore, this gene is expressed mainly during embryogenesis of cell lines typically associated with DGS developmental defects. The same workers39 then mapped UFD1L to 22q11.2 and described its structure and expression in the mouse and man. This, however, was still no more than strong circumstantial evidence.

The transcription factor dHAND is required for the survival of neuroectodermal cells in the branchial arches, and this is down-regulated in mice with the phenotypic features of DGS. The gene for dHAND, however, does not map to the DGCR. The UFD1L gene is dependent on dHAND and was also found to be down-regulated in hearts with absent dHAND transcription factor. A major breakthrough occurred when Yamagishi et al.36 managed to show that UFD1L was deleted in all 182 of their patients with DiGeorge syndrome. They went on to show that UFD1L is expressed in branchial arches I to IV, in palatal and frontonasal embryological precursors, the developing ear, and the hippocampus (where it may play a role in the learning difficulties experienced by DGS patients). Within the heart, UFD1L was expressed in the conotruncus and the IVth embryological aortic arch, where its absence would lead to the development of an interrupted aortic arch, a characteristic lesion of the DiGeorge syndrome.

Further strong corroborative evidence was found when the group found a de novo monoallelic deletion of 3 exons of the UFD1L gene in an individual with features of the DiGeorge syndrome. This patient also had a small deletion in an adjacent gene (CDC45L) but had no other deletions of the 22q11.2 region.

This association of the UFD1L gene with DGS was strengthened very recently when Lindsay et al.40 managed to produce, by genetically engineering a UFD1L deletion, a mouse model of some of the typical cardiac defects. Interestingly, however, the deleted mice only had abnormalities associated with defective development of the IVth branchial arch, but none had any of the noncardiological features associated with DGS, viz, hypocalcemia, T-cell deficiency, and thymic hypoplasia. The cardiac defects were eliminated by genetic correction of the UFD1L deletion.

CONCLUSION

These most recent developments would appear to be very strong evidence for and an elegant explanation of the role of a single gene in the pathogenesis of this important congenital disease. No other candidate gene of the DGCR has been shown to be involved so clearly in the mechanisms controlling the embryogenesis of those structures typically affected in the DiGeorge syndrome.

Some questions remain, however. What is the role of the other genes deleted in the DGCR? Does UFD1L somehow regulate their expression? They may modify the phenotype, creating further variability, or they may be involved in the regulation of UFD1L expression. What is the etiology of the anomalies found in patients without any deletions of chromosome 22, but expressing typical features of DiGeorge syndrome? Most importantly: what is the product of UFD1L and how does it influence ubiquitination and embryogenesis? Until all genes in the DGCR have been sequenced and their expression and products characterized, it appears that UFD1L will remain as the most likely candidate gene.