The effective prevention, diagnosis and management of diseases presuppose scientifically rigorous clinical investigation. This goal is difficult if the object of study is a disease of such rare frequency that few patients are available for study at any one medical center. Statistically valid comparisons in this setting are difficult and often impossible. Multicenter trials are a valid approach toward resolving this problem. The Children’s Oncology Group is an excellent example of a longstanding network that investigates best treatment of childhood cancers [3
]. This collaborative, multi-institutional approach has markedly improved the outcome of these children and affords a model that is worthy of emulation for other investigations. Fundamental information is lacking for the rare diseases that are the investigative targets of the 10 consortia that compose the RDCRN. Thus, little is known about incidence, prevalence, morbidity, mortality or response to therapy. Hence, the longitudinal studies of the UCDC and the other consortia seek to gather such information in order to provide a rational basis for subsequent clinical trials to improve outcome of conditions that commonly are devastating to patients and families and extremely costly to society in terms of the consumption of health care resources. In addition, the UCDC is also conducting other studies to answer specific questions about UCD. These include research on liver disease in AL deficiency and functional magnetic resonance imaging and spectroscopy to study brain changes in response to ammonia toxicity. These studies and other information about the UCDC are described in the following web site: http://rarediseasesnetwork.epi.usf.edu/ucdc/index.htm
As demonstrated by these results, support from federal and private philanthropic sources enabled the UCDC to expand from 5 to 8 participant institutions, with imminent plans to bring the total to 14 institutions, 2 of them in foreign sites (Toronto and Zurich). By adding sites from diverse geographic and ethnic regions, we increase the statistical power of the study cohort and lessen the probability of biasing results with an excessively narrow focus.
We found that a relatively small fraction of eligible patients (~ 27%) enrolled in the longitudinal study. This conclusion is supported by the report of the NUCDF that about a third of member families chose to participate. Although this enrollment rate is currently the highest among the RDCRN consortia [4
], we are considering strategies to improve enrollment, including changes to the contact registry, engagement with newborn screening programs and enhanced NUCDF advocacy efforts.
Thus far, the distribution of frequency among enrolled and registered UCD patients appears to be consistent with previous estimates [5
]. OTC deficiency is by far the most frequent among the UCD, followed by AL deficiency (argininosuccinic aciduria) and AS deficiency (citrullinemia). It is expected that the proportional frequency of these latter two disorders will increase in the near future as infants diagnosed by expended newborn screening using tandem mass-spectrometry [7
] are enrolled, while several other UCD, especially CPS1 and OTC deficiency cannot be identified by this screening method. Upon final analysis of this study, diagnosis by newborn screening will need to be considered as a confounding factor. Many of patients ascertained by this highly sensitive screening are asymptomatic and may remain so for many years and possibly throughout life. Thus, expanded newborn screening will likely change the natural history of screened UCD as it has done in other screened conditions [8
A second source of bias appears among patients with OTC (Figure 3). We believe that the proportion of patients with neonatal presentation should be at least equal to those with late onset disease [2
]. However, here the proportion of neonatal type patients is much smaller. This can be accounted for by patients who died before being enrolled and/or those who underwent liver transplantation soon after their initial presentation. If this hypothesis is correct, as the study gradually enrolls most patients soon after their first clinical presentation, the proportion of neonatal cases should increase. We are currently performing a retrospective analysis of data from deceased patients with UCD to factor in this bias of selection on study parameter analysis. Since most patients with OTC deficiency are apparently asymptomatic heterozygous females, it would be interesting if they show subtle deficiencies on neurocognitive testing, as have been suggested previously [9
Although still preliminary, we observed a marked difference between the number of African American and Latino participants who enrolled. While this could reflect a true difference in disease incidence, it is more likely to be related to disparity in access to medical care and/or targeted advertising of the study and/or mistrust of research due to past abuses [10
]. We plan to study this observation further and to increase our efforts to identify and reach under-represented minorities with UCD.
As expected, the ammonia scavenger drug used most frequently in chronic therapy of UCD is oral Na-phenylbutyrate (Bupheny) [11
], with Na-benzoate being far less common. L-citrulline (for OTC and CPS deficiency) and L-arginine (for AS and AL deficiency) are used frequently, although not universally. Future analysis will correlate chemical treatment modality with disease severity and other parameters for which information is collected.
Baseline assessment upon enrollment includes self reporting and medical record review with respect to neurodevelopmental parameters, as well as on patient/family perceived triggers of acute episodes of hyperammonemia. A significant proportion of enrolled patients reported developmental disabilities with more than one third reporting intellectual disability, and a similar proportion reported learning disabilities. The effects of recurrent hyperammonemia are known to cause irreversible damage to the brain [12
], resulting in the effects reported here. On the other hand, autism and mood disorders have been rarely reported [9
], as confirmed by these results. We found a considerably higher rate of developmental disabilities in patients with AL deficiency compared to OTC deficient patients. Although this could merely represent a bias of selection in enrolling a larger proportion of neonatal (severe) cases with AL than with OTC deficiency, it could represent toxic effect of disease metabolites other then ammonia (e.g. argininosuccinic acid) on the brain.
More than half of the enrolled patients had normal neurological examination, while others had a variety of abnormalities such as tone and reflex abnormalities or abnormal involuntary movements. Future analysis will correlate neurocognitive findings with various other factors to try and identify the major risk factors for developing these abnormalities.
The patients and/or their families have identified several triggers that preceded acute hyperammonemic episodes. Not surprisingly, an intercurrent infection was the most frequently cited trigger, followed by a recent change in diet. There were several dozen other triggers listed, however, none of them were implicated in an appreciable frequency.
Most patients expectedly received a protein-restricted diet in order to minimize the production of ammonia from dietary nitrogen. Diet therapy is essential, but in most instances it must be complemented by the use of ammonia scavengers, such as Na-phenylbutyrate (Buphenyl), which has substantially reduced mortality [13
]. This agent is especially effective in patients with proximal defects such as deficiencies of OTC and CPS. Patients with distal disorders (AS and AL deficiency) commonly benefit from treatment with both Na-phenylbutyrate and high doses of L-arginine [14
]. Surprisingly, arginine therapy was not utilized in almost half of the patients with AS deficiency and about one fourth of those with AL deficiency. This observation may represent the inclusion of milder or asymptomatic cases, such as those diagnosed by newborn screening, or the finding may reflect reduced compliance, non-standard care or both.
These cross-sectional data yielded important insights into the biochemical and pharmacological markers that characterize the UCD. Subjects with a deficiency of either CPS1 or OTC and who presented as newborns had low blood citrulline levels, reflecting the severity of the biochemical block. Almost all patients manifested diminished blood levels of branched-chain amino acids (BCAA) but following treatment with Na-phenylbutyrate, as previously reported [15
]. Other essential amino acids were unaffected. The decrement of blood BCAA may also result from dietary protein restriction, but other indicators of protein insufficiency -including total protein, albumin, and prealbumin – usually were normal. Similarly, citrulline treatment correlated with decreased BCAA, probably reflecting the fact that patients with a deficiency of OTC typically receive both Na-phenylbutyrate and citrulline. Not surprisingly, blood levels of major nitrogen carriers, such as glutamine and alanine, were significantly decreased in individuals who received these agents. As observed previously [16
], patients with proximal urea cycle disorders (CPS1 and OTC deficiency) display higher plasma levels of glutamine than those with distal disorders (AS, AL and ARG deficiency), probably reflecting the sequestration of waste nitrogen in citrulline, argininosiccinate or arginine, respectively.
Multicenter collaborative clinical research of a group of inborn errors of metabolism, such as UCD, allows for the first time insights into the natural history and clinical practice of these disorders. In spite of many challenges to enroll patients and potential bias of selection, the information that has been already garnered and future prospective data, are likely to markedly improve our understanding of these devastating disorders and offer better approaches to their treatments. The main benefits from this effort are yet to be realized. This will likely occur only after years of prospective study of enrolled patients, with the full participation of many clinical centers.