The effect of blood phenylalanine concentration on Kuvan™ response in phenylketonuria.

Phenylketonuria (PKU) is caused by mutations in the phenylalanine hydroxylase gene (PAH) with consequent elevation of blood phenylalanine (Phe), reduction in tyrosine (Tyr) and elevation of Phe/Tyr ratio (P/T). Although newborn screening for PKU with early dietary treatment improved severe, irreversible brain damage, older patients suffer reversible losses in executive function when Phe concentrations are elevated. The maintenance of strict nutritional control in older children and adults remains difficult. An adjunct to dietary therapy, oral tetrahydrobiopterin (BH(4)) has recently been approved by the Food and Drug Administration as a stable, synthetic BH(4) called Kuvan™. Published studies of Kuvan response in PKU varies and involved primarily children. In this prospective study we evaluated dose-response, response frequency and factors predicting response in 21 patients with PKU (aged 8-30 years), who required life-long dietary treatment. Response to Kuvan was defined at 24h (acute) and over 4 weeks (chronic) as a ≥ 30% decline in the Phe or P/T ratio. A dose of 20mg/kg Kuvan was chosen with 29% responding in 24h and 33% of patients at 4 weeks. We then compared baseline Phe, Tyr, P/T, Phe intake, and frequency of "severe" versus "moderate" mutant PAH alleles among acute and chronic responders and non-responders to Kuvan. Predictors of response to Kuvan, both acute and chronic were baseline Phe and baseline P/T. Baseline Phe and P/T were higher among non-responders (P<0.05). By contrast baseline Tyr was similar (P=0.45). Phe intake tended to be higher (18 ± 20mg/kg/24h) among Kuvan responders than non-responders (15 ± 11 mg/kg/24h), P<0.07 NS. Similarly the frequency of "severe" mutant PAH alleles tended to be more frequent (67%) among non-responders than responders (40%) by Chi(2) test, P=0.08 NS. These results were reproducible in a "responder" to Kuvan. To assess directly the effect of elevated blood Phe, Phe was lowered in four, "non-responder" patients, but all failed to respond to Kuvan. We conclude that baseline blood Phe and P/T ratio can predict increased probability for response to Kuvan by patients with classic PKU, but the in vivo mechanisms of response to Kuvan remain enigmatic.

Galactose toxicity in animals.

In most organisms, productive utilization of galactose requires the highly conserved Leloir pathway of galactose metabolism. Yet, if this metabolic pathway is perturbed due to congenital deficiencies of the three associated enzymes, or an overwhelming presence of galactose, this monosaccharide which is abundantly present in milk and many non-dairy foodstuffs, will become highly toxic to humans and animals. Despite more than four decades of intense research, little is known about the molecular mechanisms of galactose toxicity in human patients and animal models. In this contemporary review, we take a unique approach to present an overview of galactose toxicity resulting from the three known congenital disorders of galactose metabolism and from experimental hypergalactosemia. Additionally, we update the reader about research progress on animal models, as well as advances in clinical management and therapies of these disorders.

Screening newborns for galactosemia using total body galactose oxidation to CO2 in expired air.

Classic galactosemia is caused by impaired galactose-1-phosphate uridyltransferase (GALT EC 2.7.712). If discovered and treated within the first days of life, the acute problems of hepatocellular damage, sepsis, and death are prevented. However, chronic problems such as ataxia, tremor, dyspraxic speech, and ovarian failure may occur. To determine whether screening newborns before discharge from the nursery for GALT deficiency is feasible and whether acute and chronic signs could be prevented by earlier intervention, we developed a simplified "breath test." We quantitated total body oxidation of C-D-galactose to CO2 in expired air by normal newborns between 2 h and 2 mo of age and compared their results to older children with GALT deficiency. We found no differences in total body galactose oxidation (TBGO) among normal newborns up to 48 h of age, but a 2-fold rise in TBGO developed during their first 2 wk of life. Older children with galactosemia had significantly less oxidative capacity than normal newborns. We conclude that newborn breath testing for total body galactose oxidation is feasible before discharge from nursery. It has potential utility for both preventing acute neonatal toxicity and determining the mechanisms producing long-term complications such as ovarian failure, dyspraxia, ataxia, and tremors.

Detection of pathogenic gene copy number variations in patients with mental retardation by genomewide oligonucleotide array comparative genomic hybridization.

Genomic imbalance is a major cause of developmental disorders. Microarray-based comparative genomic hybridization (aCGH) has revealed frequent imbalances associated with clinical syndromes, but also a large number of copy number variations (CNVs), which have complicated the interpretation of results. We studied 100 consecutive patients with unexplained mental retardation and a normal karyotype using several platforms of CGH arrays. A genomewide array with 44,290 oligonucleotide probes (OaCGH44K) detected imbalances in 15% of cases studied with sizes ranged from 459 kb to 19 Mb while revealing a small number of CNVs (0.72/individual). Another platform with approximately 240,000 oligonucleotide probes (OaCGH244K) revealed a large number of CNVs (20/individual) in selected cases and their normal parents. We used a comprehensive approach for interpreting the results of aCGH, including consideration of the size, inheritance and gene content of CNVs, and consultation with an online Database of Genomic Variants (DGV) and Online Mendelian Inheritance in Men (OMIM) for information on the genes involved. Our study suggests that genomewide oligonucleotide arrays such as the OaCGH44K platform can be used as a powerful diagnostic tool for detection of genomic imbalances associated with unexplained mental retardation or syndromic autism spectrum disorders. It is interesting to note that a small number of common variants were revealed by OaCGH244K in some study subjects but not in their parents and that some inherited CNVs had altered breakpoints. Further investigations on these alterations may provide useful information for understanding the mechanism of CNVs.

Prevention of a molecular misdiagnosis in galactosemia.

PURPOSE: The polymerase chain reaction is generally used for mutational analysis of the galactose-1-phosphate uridyl transferase (GALT) gene in the diagnosis of galactosemia. This method is problematic when used in families of Ashkenazi Jewish descent. METHODS: We amplified the GALT gene from leukocyte DNA followed by allele specific oligonucleotide hybridization, DNA sequencing and Southern Blot analysis to determine the mutant alleles causing galactosemia in a representative Jewish family. RESULTS: The proband's diagnosis of galactosemia was confirmed by high levels of erythrocyte galactose-1-phosphate, absence of erythrocyte GALT activity and impaired total body oxidation of galactose to expired CO2. Initial molecular analysis of GALT alleles in the family showed homozygosity for a K285N missense mutation in the proband, homozygosity for N314D in the mother and heterozygosity for N314D and K285N in the father. These results contradicted Mendelian logic. Southern blot hybridization with GALT cDNA proved the presence of a complex 5 kb GALT deletion in the proband and her mother's DNA enabling a corrected genotype. CONCLUSIONS: Since a deletion of the GALT gene is a common mutation causing galactosemia among Ashkenazim Jewish families, this deletion should be suspected and tested for by genomic hybridization or by using primers specific for the 5 kb deletion.

The history of the SIMD: from small molecules to metabolomics.

The Society for Inherited Metabolic Disorders (SIMD) recently completed its 28th annual meeting and requested that its birth and development be documented. Accordingly, this communication reviews the origins of the SIMD and considers its development over the past three decades. Each decade had growth and differentiation regarding policy making, international connections, highlights and tragedies. Past contributions are defined and future directions for the SIMD are subjected to prophesy.

A structure-function study of MID1 mutations associated with a mild Opitz phenotype.

The X-linked form of Opitz syndrome (OS) affects midline structures and produces a characteristic, but heterogeneous, phenotype that may include severe mental retardation, hypertelorism, broad nasal bridge, widow's peak, cleft lip/cleft palate, congenital heart disease, laryngotracheal defects, and hypospadias. The MID1 gene was implicated in OS by linkage to Xp22. It encodes a 667 amino acid protein that contains a RING finger motif, two B-box zinc fingers, a coiled-coil, a fibronectin type III (FNIII) domain, and a B30.2 domain. Several mutations in MID1 are associated with severe OS. Here, we describe an intelligent male with a milder phenotype characterized by hypertelorism, broad nasal bridge, widow's peak, mild hypospadias, pectus excavatum, and a surgically corrected tracheo-esophageal fistula. He has an above average intelligence and no cleft lip/palate or heart disease. We identified a novel mutation in MID1 (P441L) which is in exon 8 and functionally associated with the FNIII domain. While OS phenotypes have been attributed to mutations in the C-terminal part of MID1, little is currently known about the structure-function relationships of MID1 mutations, and how they affect phenotype. We find from a literature review that missense mutations within the FNIII domain of MID1 are associated with a milder presentation of OS than missense mutations elsewhere in MID1. All truncating mutations (frameshift, insertions/deletions) lead to severe OS. We used homology analysis of the MID1 FNIII domain to investigate structure-function changes caused by our missense mutation. This and other missense mutations probably cause disruption of protein-protein interactions, either within MID1 or between MID1 and other proteins. We correlate these protein structure-function findings to the absence of CNS or palatal changes and conclude that the FNIII domain of the MID1 protein may be involved in midline differentiation after neural tube and palatal structures are completed.

Cystathionine beta-synthase deficiency: effects of betaine supplementation after methionine restriction in B6-nonresponsive homocystinuria.

PURPOSE: For treatment of cystathionine beta-synthase (CbetaS) deficiency, we determined the effect of betaine (N,N,N-trimethylglycine) therapy and examined the genotype-phenotype relationships to betaine. METHODS: In five patients with B6-nonresponsive homocystinuria, we defined the CbetaS genotypes and determined metabolic responses to betaine as an additive to traditional dietary methionine restriction. RESULTS: After betaine therapy, tHcy declined (mean 47.4 micromol/L; range: -21.2 to -104.0 micromol/L; P=0.02), whereas total plasma cysteine and methionine did not change. Plasma methionine/tHcy ratios increased by 5.45 (range: +1.5 to 15.3; P=0.05) inpatients with B6-nonresponsive alleles. CONCLUSION: Betaine improves metabolic control in B6-nonresponsive patients with homocystinuria after optimum dietary control.

Iron status of children with phenylketonuria undergoing nutrition therapy assessed by transferrin receptors.

PURPOSE: The purpose of the study was to determine the incidence of iron deficiency in children undergoing therapy for phenylketonuria using serum transferrin receptor and ferritin concentrations. METHODS: A 1-year study was conducted in 37 children 2 <13 years old with phenylketonuria (8 fed Periflex [Group I], 15 fed Phenex-2 Amino Acid-Modified Medical Food [Group II], and 14 fed Phenyl-Free [Group III]). Hemoglobin, hematocrit, serum transferrin receptor, and ferritin concentrations were assessed at baseline and 12 months and intakes of protein, iron, and vitamin C were evaluated at baseline and at 3-month intervals. Transferrin receptor and ferritin concentrations were analyzed for group differences by analysis of variance. RESULTS: Mean protein, iron, and vitamin C intakes of all 3 groups of children were greater than Recommended Dietary Intakes for age. Only 2 of 60 3-day diet diaries of Group II children failed to contain 100% of Recommended Dietary Intakes for iron during study. The following number of children had iron status indices outside reference ranges at study end: 7 children, transferrin receptor/ferritin ratios; 4 children, transferrin receptors; 2 children, hematocrit; 1 child, ferritin. No correlation was found between iron intake and any index of iron status. CONCLUSIONS: The transferrin receptor/ferritin ratio appeared to be the most sensitive index of iron deficiency in this study. Reasons for iron deficiency with greater than recommended iron intakes by children with phenylketonuria may be multiple. Early assessment and therapy of iron deficiency may improve cognitive and behavioral outcomes of children with PKU.

Verbal dyspraxia and galactosemia.

Classical galactosemia is an autosomal recessive disorder resulting from deficient galactose-1-phosphateuridyl transferase (GALT) activity. Verbal dyspraxia is an unusual outcome in galactosemia. Here we validated a simplified breath test of total body galactose oxidation against genotype and evaluated five potential biochemical risk indicators for verbal dyspraxia in galactosemia: cumulative percentage dose (CUMPCD) of (13)CO(2) in breath, mean erythrocyte galactose-1-phosphate, highest erythrocyte galactose-1-phosphate, mean urinary galactitol, and erythrocyte GALT activity. Thirteen controls and 42 patients with galactosemia took a (13)C-galactose bolus, and the (CUMPCD) of (13)CO(2) in expired air was determined. Patients with <5% CUMPCD had mutant alleles that severely impaired human GALT enzyme catalysis. Patients with > or =5% CUMPCD had milder mutant human GALT alleles. Twenty-four patients consented to formal speech evaluation; 15 (63%) had verbal dyspraxia. Dyspraxic patients had significantly lower CUMPCD values (2.84 +/- 5.76% versus 11.51 +/- 7.67%; p < 0.008) and significantly higher mean erythrocyte galactose-1-phosphate (3.38 +/- 0.922 mg/dL versus 1.92 +/- 1.28 mg/dL; p = 0.019) and mean urinary galactitol concentrations (192.4 +/- 75.8 mmol/mol creatinine versus 122.0 +/- 56.4; p = 0.048) than patients with normal speech. CUMPCD values <5%, mean erythrocyte galactose-1-phosphate levels >2.7 mg/dL, and mean urinary galactitol levels >135 mmol/mol creatinine were associated with dyspraxic outcome with odds ratios of 21, 13, and 5, respectively. We conclude that total body oxidation of galactose to CO(2) in expired air reflects genotype and that this breath test is a sensitive predictor of verbal dyspraxia in patients with galactosemia.

Gaucher disease: in vivo evidence for allele dose leading to neuronopathic and nonneuronopathic phenotypes.

Gaucher disease, a common lysosomal storage disorder, is associated with mutations at the acid beta-glucosidase (GCase) locus. Two affected individuals are described to share a common mutant allele, but manifest different clinical categorical phenotypes. A 57-year-old female, with Gaucher disease type 1 and Cherokee ancestry, was homozygous for a rare mutant allele encoding Lys79Asn (K79N). A 2-year-old Caucasian male, with Gaucher disease type 3 and Cherokee ancestry, was a heteroallelic homozygote for this same allele (K79N) and a novel complex mutation (null allele). The shared alleles were identical as determined by complete gene sequencing, suggesting a founder effect. The discrepant phenotypes (types 1 and 3) in these two patients provide support for a threshold of residual activity necessary to "protect" the central nervous system (CNS) from the pathogenic effects of Gaucher disease, indicating an allele dose-effect. Designation of genotype associations with specific phenotypes must be assessed with this perspective.

Characterization of a carbohydrate response element regulating the gene for human galactose-1-phosphate uridyltransferase.

Human galactose-1-phosphate uridyltransferase (hGALT) is a central enzyme in the conserved pathway by which galactose is converted to energy, UDP-galactose and UDP-glucose. A natural mutation that deleted -119 to -116 bp (delGTCA) of the promoter decreased hGALT mRNA and enzyme activity and prompted analysis of hGALT gene regulation. Regulatory domains were identified by inspection and confirmed in a reporter system. Previous studies by others were confirmed that HepG2 cells grown in D-glucose increased hGALT enzyme activity and mRNA by 30%. We extended these observations by sequencing the promoter region and identifying a potential carbohydrate response element (ChoRE). The response to glucose rose to 190% when a plasmid construct containing a luciferase reporter and only the -165 bp region as a promoter was transfected into HepG2 and NIH:OVCAR-3. By contrast, fibroblasts transfected with the identical construct failed to respond to glucose. Within the -165 bp region there were two enhancer (E-box) motifs that encompassed the delGTCA mutation. The deletion diminished the positive regulatory response, but an additional GTCA repeat unexpectedly increased the response. Using this postulated ChoRE as a probe in electrophoretic mobility shift assays, multiple nuclear proteins bound and one was identified as a member of the basic/helix-loop-helix/leucine zipper enhancer-binding (b/HLH/LZ) family. Increased binding of proteins correlated with increased hGALT expression when the spacing between E-box motifs was enlarged but the carbohydrate response was dampened. When the 3(')E-box was mutated, b/HLH/LZ binding and gene expression were abolished. We conclude that the hGALT promoter region contains a ChoRE in which the spacing between and the sequence of its E-box motifs are critical. One nuclear protein of the b/HLH/LZ family is necessary, but not sufficient for the carbohydrate response.

Cessation of enzyme replacement therapy in Gaucher disease.

PURPOSE: Enzyme replacement therapy (ERT) is a promising therapeutic intervention for lysosomal storage diseases. Posttranslationally engineered human beta-glucocerebrosidase (Ceredase/Cerezyme) is commercially available and is the standard ERT for Type I Gaucher disease. Cessation of therapy is sometimes necessary for personal or financial reasons, but the consequences of discontinuation are unknown. This study reports results of discontinuing therapy in four patients with Type I Gaucher disease with different genotypes and varying degrees of clinical involvement. METHODS: Patient genotypes were as follows: N370S/L444P (Patients 1 and 2), K79N/K79N (Patient 3), and N370S/N370S (Patient 4). All were evaluated before, during, and after withdrawal from ERT. Patients 1, 2, and 3 were studied after reinstituting ERT. The following parameters were documented at 3- to 12-month intervals in all patients: hemoglobin, platelet count, angiotensin-converting enzyme, spleen volume, liver volume, femoral magnetic resonance imaging, bone density, and urinary pyridinium crosslinks. RESULTS: After cessation of therapy, Patients 1, 2, and 3 had more dramatic regression in hematological and visceral parameters than Patient 4 and required reinstitution of ERT within 2 years. All three patients recovered posttreatment status within 4 years of reinstituting ERT. Patient 4 remained stable 6 years after cessation of ERT. CONCLUSIONS: Regression of disease status in patients with Type I Gaucher disease after cessation of ERT conformed to the genotype-phenotype relationships of disease onset. Careful monitoring and reinstitution of ERT enabled previously attained treatment status.