Standardising clinical outcomes measures for adult clinical trials in Fabry disease: A global Delphi consensus.

BACKGROUND: Recent years have witnessed a considerable increase in clinical trials of new investigational agents for Fabry disease (FD). Several trials investigating different agents are currently in progress; however, lack of standardisation results in challenges to interpretation and comparison. To facilitate the standardisation of investigational programs, we have developed a common framework for future clinical trials in FD. METHODS AND FINDINGS: A broad consensus regarding clinical outcomes and ways to measure them was obtained via the Delphi methodology. 35 FD clinical experts from 4 continents, representing 3389 FD patients, participated in 3 rounds of Delphi procedure. The aim was to reach a consensus regarding clinical trial design, best treatment comparator, clinical outcomes, measurement of those clinical outcomes and inclusion and exclusion criteria. Consensus results of this initiative included: the selection of the adaptative clinical trial as the ideal study design and agalsidase beta as ideal comparator treatment due to its longstanding use in FD. Renal and cardiac outcomes, such as glomerular filtration rate, proteinuria and left ventricular mass index, were prioritised, whereas neurological outcomes including cerebrovascular and white matter lesions were dismissed as a primary or secondary outcome measure. Besides, there was a consensus regarding the importance of patient-related outcomes such as general quality of life, pain, and gastrointestinal symptoms. Also, unity about lysoGb3 and Gb3 tissue deposits as useful surrogate markers of the disease was obtained. The group recognised that cardiac T1 mapping still has potential but requires further development before its widespread introduction in clinical trials. Finally, patients with end-stage renal disease or renal transplant should be excluded unless a particular group for them is created inside the clinical trial. CONCLUSION: This consensus will help to shape the future of clinical trials in FD. We note that the FDA has, coincidentally, recently published draft guidelines on clinical trials in FD and welcome this contribution.

Proposed high-risk screening protocol for Fabry disease in patients with renal and vascular disease.

Fabry disease is a complex, multisystemic and clinically heterogeneous disease with prominent urinary excretion of globotriaosylceramide (Gb(3)), the principal substrate of the deficient enzyme, alpha-galactosidase A. Some measure of specific treatment is possible with enzyme replacement therapy, which can be applied safely and effectively to Fabry patients. Incidence estimations of Fabry disease vary widely from 1:55 000 to 1:3000 male births. The true incidence is likely to be higher than originally thought, owing to the existence of milder variants of the disease. The main complications of Fabry disease are a 100-fold increased risk of ischaemic stroke, cardiac disease, a wide variety of arrhythmias, valvular dysfunction and cardiac vascular disease, as well as progressive renal failure usually associated with significant proteinuria. These clinical manifestations are non-specific and are often mistaken for symptoms of other disorders, thus complicating the confirmation of diagnosis. Other clinical features of the disease are often absent (angiokeratoma), subtle (corneal opacities and hypohidrosis), or unaccompanied by specific physical findings (acroparaesthesias) indicating the true nature of the underlying disease. We propose the hypothesis that alpha-galactosidase A deficiency is a modifiable cardiovascular risk factor in the general population. This hypothesis may be tested by a non-invasive high-risk screening protocol for Fabry patients with ischaemic strokes and a variety of cardiac, and renal complications. These patients would benefit from diagnosis, appropriate treatment, follow-up and surveillance. Early detection of Fabry patients would also benefit affected relatives, many of whom do not have a clear diagnosis of their clinical condition.

Quebec neonatal mass urinary screening programme: from micromolecules to macromolecules.

The Quebec Mass Urinary Screening Programme, initiated in 1971, has resulted in the screening of more than 2,500,000 newborns in the province of Quebec for 25 inherited Mendelian disorders divided into two groups. The first group concerns urea cycle disorders (citrullinaemia, hyperargininaemia, argininosuccinic aciduria), ketotic hyperglycinaemia, and organic acidurias (methylmalonic aciduria, glutaric aciduria type I, etc.); the second group relates to disorders of amino acid metabolism (cystathioninuria, prolidase deficiency, etc.) and transport (Fanconi syndrome, cystinurias, Hartnup syndrome, etc.). The main goal of the Programme is to detect and prevent these genetic diseases, some detectable only in urine, before the onset of clinical symptoms. A multiplex thin-layer chromatography methodology was developed, in which metabolites in urine are resolved and visualized by the sequential application of four different reagents to detect aminoacidopathies and organic acidurias. The technique is simple, reproducible, inexpensive and rapid, allowing the analysis of 500 samples daily by a single technician. The voluntary compliance of the parents is excellent, averaging 90% per year. Over the years, we have established a dynamic process, developing techniques or new reagents to detect as many treatable disorders as possible, now evaluating macromolecules associated with lysosomal storage disorders, mainly globotriaosylceramide (Gb3) for Fabry disease. We present here the methodology, infrastructure in place, results and recent statistics of the well-established Quebec Mass Urinary Screening Programme. We also report a study by tandem mass spectrometric analysis of urinary Gb3 in Fabry disease for the follow-up and monitoring of Fabry patients, as well as for its possible application to mass and high-risk screening programmes.

Development of a filter paper method potentially applicable to mass and high-risk urinary screenings for Fabry disease.

Fabry disease is an X-linked lysosomal storage disorder of glycosphingolipid catabolism resulting from a deficiency of the enzyme alpha-galactosidase A, and leading to the progressive accumulation of one biomarker, globotriaosylceramide (Gb(3)), predominantly elevated in the urine of these patients. We have developed a technique for the analysis of total Gb(3) in urine samples collected on filter paper, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a triple quadrupole instrument. Existing Gb(3) techniques being both time- and labour-intensive, this filter paper method eliminates lipid extraction, glycolipid isolation, centrifugation and evaporation steps, while maintaining sensitivity and efficiency. The stability of Gb(3) on filter paper was good for a 7-week period under different temperature conditions. Normal control values were established and the technique was tested with anonymized samples from Fabry hemizygotes and heterozygotes. The levels of total Gb(3) in all classical hemizygotes were well above the control values and all heterozygotes, except two nonexcretors, were above the reference level. The proposed novel filter paper method favours the collection, storage and shipment of samples. It is simple and efficient for a feasibility study, potentially applicable to the determination of total urinary Gb(3) in the newborn population as part of a screening programme, and could also be used in high-risk screening laboratories. Since the incidence of Fabry disease is hard to establish, owing to the heterogeneous clinical expression of the visible phenotype, this feasibility study could help determine its actual incidence in the Quebec population.

Screening for neuroblastoma at 3 weeks of age: methods and preliminary results from the Quebec Neuroblastoma Screening Project.

A large neuroblastoma screening study was recently started in the province of Quebec, Canada. This project, a collaboration between the Quebec Network for Genetic Medicine and the University of Minnesota, is studying the impact of screening infants for the preclinical detection of neuroblastoma on the population-based mortality caused by this tumor. All infants born in Quebec during a 5-year period will be screened twice, at 3 weeks and at 6 months. Urinary homovanillic acid and vanillylmandelic acid determination from dried filter paper samples is used for screening. Initial qualitative screening is done by means of thin-layer chromatography with confirmatory quantitative screening by gas chromatography-mass spectrometry (GC-MS). During the initial 6 months of 3-week screening, 41,673 neonates (92% compliance rate) were screened and 10.6% of them were tested also by GC-MS. Nine of these neonates had positive results on two GC-MS tests and were referred for evaluation to rule out the presence of neuroblastoma. Four had the tumor, 1 had a calcified adrenal gland, and 4 had no tumor detected. Three additional neonates had clinical diagnosis of neuroblastoma before they reached the screening age of 3 weeks. A neuroblastoma that did not secrete homovanillic acid or vanillylmandelic acid was diagnosed clinically in 1 additional patient who tested negative by screening.

An automated method for the determination of orotic acid in the urine of children being screened for metabolic disorders.

Urinary orotic acid is believed to be a valuable probe for early diagnosis of inborn errors of metabolism leading to hyperammonemia and increased pyrimidine synthesis. For the purpose of our urinary mass screening programme, we developed an automated colorimetric method which is reliable in the range of 1 to 50 micrograms/ml orotic acid and allows analyses at a rate of 160 samples per hour. Preliminary results are presented which illustrate that various disorders can be recognized by measuring orotic acid in urine.

Rapid thin-layer chromatographic method for the detection of urinary methylmalonic acid.

1. Simple and rapid thin-layer and micro-thin-layer chromatography techniques are described for the detection of methylmalonic acid in urine. 2. The separation of methylmalonic acid from a crude urine sample is performed by thin-layer chromatography with a mixture of silica gel-cellulose and butanol - acetic acid - water solvent system. The methylmalonic acid spot is visualized with tetrazotized o-dianisidine. 3. This system has been successfully developed for a urinary screening programme; it was shown as simple, convenient and rapid, eliminating false-positives and allowing the detection of even traces of methylmalonic acid.

Simple and rapid system for screening and identification of reducing sugars in urine.

As part of our screening programme for metabolic disorders we needed a rapid, simple, inexpensive means to detect reducing sugars in urine, with a simple but precise test for their identification. Our system comprises a bismuth reduction test for reducing sugars, with unidimensional thin layer chromatography on silica gel and color development with diphenylamine--aniline for definitive identification.

Diet and medications giving positive ninhydrin reactions on TLC in a newborn urinary screening program.

During amino acid analysis of over 520,000 urine samples (provided by a urinary screening program), we often noticed different dietary compounds or medications giving positive ninhydrin reactions which could interfere with the interpretation of the thin layer chromatograms. Consequently, we have done an in vitro study of the chromatographic behaviour of the most frequently encountered artifacts in urine. We believe that the results of this work should make it easier for other laboratories doing similar analyses to recognize the presence of such compounds, thus facilitating the evaluation of their chromatograms.

Determination of urinary homovanillic and vanillylmandelic acids from dried filter paper samples: assessment of potential methods for neuroblastoma screening.

Urinary homovanillic (HVA) and vanillylmandelic (VMA) acids were analyzed on 200 random urine samples from patients with neuroblastoma and controls, after the samples had been dried onto absorbent filter paper. The acids were determined quantitatively by gas chromatography (GC) and qualitatively by thin layer chromatography (TLC). The results were analyzed for correlation between liquid urine samples and urine dried on filter paper and between TLC and GC methods. A high overall correlation for HVA and VMA (99%) was found between liquid and dried filter samples analyzed by GC. The correlations were more significant for samples with elevated levels of these acids than for those with normal levels. Normalization of the results to the urinary creatinine concentration (UCr) is indicated due to variations in urine concentration. Results from TLC analysis showed a false positive rate of 3.5% and a false negative rate of 0.5% compared to GC analysis. This work suggests that a combination of a sensitive TLC method with a rapid quantitative GC method would be suitable for mass neuroblastoma screening in infants.

A metabolomic study reveals novel plasma lyso-Gb3 analogs as Fabry disease biomarkers.

Fabry disease is an X-linked, multisystemic lysosomal storage disorder due to alpha-galactosidase A deficiency. It is characterized by the accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb(3)), in biological fluids, vascular endothelium, heart, and kidneys. Treatment by enzyme replacement therapy has been shown to be beneficial in both males and females affected with the disease. In addition to Gb(3), increased concentrations of globotriaosylsphingosine (lyso-Gb(3)) have recently been reported in urine and plasma of Fabry patients. The overall objective of this metabolomic study was to identify and characterize new potential plasma biomarkers in treated and untreated males and females affected with Fabry disease which might better reflect disease severity and progression. We employed a time-of-flight mass spectrometry metabolomic approach using plasma samples of Fabry patients compared to age-matched controls. We found three new lyso-Gb(3) analogs in Fabry patients presenting m/z ratios at 802, 804, and 820. As previously detected by our group, we also found a m/z ratio of 784 corresponding to the lyso-Gb(3) molecule minus two hydrogen atoms. Using exact mass measurements and tandem mass spectrometry, we confirmed that these analogs result from modifications of the lyso-Gb(3) sphingosine moiety. We evaluated the relative plasma concentration by measuring area counts for each lyso-Gb(3) analog. None of these analogs was detected in the majority of healthy controls. The relative concentration of each analog was higher in males compared to female Fabry patients. We demonstrated that mass spectrometry combined to a metabolomic approach is a powerful tool to detect and identify new potential biomarkers.

Novel gb(3) isoforms detected in urine of fabry disease patients: a metabolomic study.

Fabry disease is characterized by the accumulation of globotriaosylsphingosine (lyso-Gb(3)) and globotriaosylceramide (Gb(3)) in biological fluids and tissues. Metabolomic studies recently undertaken by our group, showed the presence of novel plasma and urine lyso-Gb(3)-related analogs in male and female Fabry patients. These analogs are distinguished by differences in structure of the sphingosine moiety. The principal aim of this study was to evaluate the possibility of detecting other Fabry disease biomarkers structurally related to Gb(3). A time-of-flight mass spectrometry metabolomic approach, focusing on mass-to-charge (m/z) ratios from 1000 to 1200 Da, was devised. This m/z window corresponds to the isoforms and potential analogs of Gb(3). Five different categories of Gb(3)- related isoforms/analogs were detected: Gb(3)-related isoforms with saturated fatty acids, methylated Gb(3)-related isoforms, Gb(3)-related isoforms/analogs with one double bond, Gb(3) analogs with hydrated sphingosine, and Gb(3)-related isoforms/analogs with two double bonds. A secondary objective was to elucidate the relationship between Gb(3) and lyso-Gb(3). The methylation observed on Gb(3)-related analogs was not detected on lyso-Gb(3). We speculate that the methylated Gb(3) may be an intermediate compound in the deacylation of Gb(3) to generate the lyso-Gb(3) molecule. We are in the process of devising a quantification methodology for these methylated Gb(3)-related analogs in Fabry patients to try to understand the underlying biochemical mechanisms involved in this complex disease.

Newborn urine screening programme in the province of Quebec: an update of 30 years' experience.

The introduction of our voluntary mass screening programme in 1971, in the province of Quebec, has permitted us to detect different inborn errors of metabolism in the newborn population using a thin-layer chromatographic (TLC) technique with sequential use of different sprays on the same plate. Abnormalities in amino acids and organic acids are detected in urine filter paper specimens of 21-day-old babies. Initial parental compliance is 90% and climbs to 99.25% for repeat sample requests. Screening is centralized in one laboratory, while diagnosis, counselling, management and follow-up are done in four regional centres. Over 25 inherited Mendelian disorders can be identified. There have been certain modifications in our programme throughout the years in order to increase efficiency, screen for a larger number of disorders, improve the quality of the collection of the urine filter paper samples, increase parental compliance and better manage the data bank. However, one goal has remained a priority: early prevention of genetic diseases. We present an overall view of our screening programme with an add-on technique to detect different organic acidurias, our recent statistics and the modifications implemented over the years.

Newborn urine screening experience with over one million infants in the Quebec Network of Genetic Medicine.

We screened urine for chemical individuality in over 1 million newborn infants, by various chromatographic (thin-layer), chemical and spectrophotometric methods, 12 procedures in all. The programme is part of the Quebec Network of Genetic Medicine. Voluntary urine screening began in 1971 and has evolved with changes in choice of tests and times of sample collection. Urine samples were collected on filter paper at either 5, 14 or 21 days after birth; results were best with the 21-day test. Compliance is over 94% with the latter and over 98% with requests for repeat samples. Screening is centralized in one laboratory; follow-up diagnosis, counselling and management are done at four regional centres. Incidence of phenotypes ranged from 1:4300 live births (for expressed cystinuria alleles) to 1 per million (for hyperargininaemia). Over 20 inherited Mendelian disorders were identified. 30 patients required aggressive medical management. We show how this programme can be used for neuroblastoma screening.

Ontogeny modifies manifestations of cystinuria genes: implications for counseling.

Among 339,868 newborn infants screened at 3 weeks of age (91% compliance rate), 730 had elevated rates of excretion of cystine and the dibasic amino acids lysine, ornithine, and arginine; 191 infants had persistent "infantile cystinuria" on follow-up screening (100% compliance). Apparent incidence of the phenotype was 562 per million infants; this rate is seven times higher than for classic cystinuria in the adult segment of the Quebec population. We studied longitudinally 26 probands 2 to 4 months of age. Initially, each excreted cystine and dibasic amino acids at much higher levels than did normal infants or either parent. From parental phenotypes (heterozygous or homozygous normal) and urine amino acid excretion values at 6 months of age in probands, the infants were classified as either heterozygous for the various classic cystinuria genotypes--type I ("silent"), eight infants; type II (high excretor), three; type III (moderate excretor), nine--or homozygous (and genetic compound), six. Urine amino acid excretion diminished steadily with age, to reach the variant parental value in heterozygous infants but not in homozygotes. Cystinuria heterozygotes, with the possible exception of some type I individuals, could not be distinguished reliably from homozygotes in early infancy, although homozygotes had significantly higher excretion values as a group. We deduce that renal ontogeny amplifies phenotypic expression of cystinuria alleles, thus influencing correct classification of genotype (heterozygote vs homozygote, and type of allele). These findings have implications for counseling and the need for follow-up of infantile cystinuria.

Feasibility of chemical screening of urine for neuroblastoma case finding in infancy in Quebec.

Neuroblastoma is the most common fatal solid tumour of childhood. Studies in Japan suggest that screening urine at 6 months for tumour-derived metabolites greatly improves early case finding and prognosis. The incidence rate of neuroblastoma in Quebec is at least 1 per 10,330 live births, higher than that of all other diseases responding to early treatment except congenital hypothyroidism screened for in the Quebec Network of Genetic Medicine. The feasibility of chemical screening of urine for elevated levels of homovanillic acid and vanillylmandelic acid in Quebec was assessed. The cost-effectiveness of screening 100,000 infants per year would be high (cost-benefit ratio 2.4), with a net saving of about $280,000 and eight lives per year. The estimated cost of adding neuroblastoma screening to the existing urine metabolite screening program is $70,700. The apparent sensitivity of the proposed test is 0.859 and the rate of false-positive results about 0.1%, both acceptable values. The attitude of potential participants toward the present urine screening program and the addition of a "tumour test" was positive. The results indicate that a pilot study of neuroblastoma screening in Quebec could be undertaken.

Outcome of individuals with low-moderate methylmalonic aciduria detected through a neonatal screening program.

BACKGROUND: The clinical spectrum of methylmalonic aciduria (MMAuria) ranges from severe, neonatal acidosis to benign asymptomatic organic aciduria. In 1975, screening for MMAuria was established in the province of Quebec. Although newborn screening programs facilitate presymptomatic detection and treatment and also detect asymptomatic variants, uncertainties about potential long-term hazards of mild to moderate elevations of MMA create concern. The objective of this study was to examine the outcome of individuals excreting low to intermediate quantities of MMA, ascertained by a newborn screening program. RESULTS AND STUDY DESIGN: One hundred and thirty-six individuals with elevations of urinary MMA were initially identified by the screening program; 122 individuals were noted to have excretion of urinary MMA <1400 micromol/mmol creatinine. At follow-up assessment at 1 year of age, in 65 of these 122 individuals, the MMA excretion had resolved. Of the remaining individuals, 9 were lost to follow-up, 13 had symptoms, and the remaining 35 were free of symptoms. Among the 35 individuals with asymptomatic persistent MMAuria, MMA excretion has resolved in 13 over 1 year; 22 individuals exhibit persistent low-moderate MMAuria (range, 210 to 1133 micromol/mmol creatinine). CONCLUSION: Follow-up examination of individuals in the latter asymptomatic cohort with persistent low-moderate MMAuria indicates normal somatic and cognitive outcomes.