Classical hemocystinuria in two Filipino patients

Classical hemocystinuria is an inborn error of metabolism caused by a deficiency of cystathionine beta-synthase that converts hemocysteine to cystathionine. This then leads to elevation of hemocysteine which results in abnormalities of the eyes, skeleton, central nervous system and vascular hemocystinuria. Patient 1 presented with lens dislocation and mental retardation while Patient 2 presented with thromboembolism, mental retardation and lens dislocation. The elevated plasma hemocysteine and methionine levels lead to the diagnosis of hemocystinuria.

Early diagnosis and specialist care in the management of congenital hypothyroidism

Background. Newborn screening for congenital hypothyroidism (CH) in the Philippines was introduced in 1996. It is universally accepted that early detection through newborn screening and timely treatment can improve the physical and neuro-cognitive development of patients. As of December 2010, the prevalence of CH is 1 in 3,324 among 2,389,959 newborns screened. Objective. We sought to evaluate the role of timing of diagnosis, compliance with treatment, and specialist care on growth and development (mental and physical) of patients with congenital hypothyroidism detected through newborn screening. Methods. Of the 326 patients identified through newborn screening between July 1996-December 2008 at the Newborn Screening Center-National Institutes of Health, 86 patients participated in the study. With the parents' or guardians' consent, general physical examination and neuro-cognitive evaluation were done; FT4 and TSH were determined. Prevalence of poor control of disease (high TSH with normal or low FT4 or normal TSH with low FT4), stunting, and cognitive delay were each estimated at 95% confidence level and the associations of early diagnosis, initial and continuing specialist care with these conditions were determined by multiple logistic regression analyses. Results. The prevalences (95% confidence interval) were: poor control of disease 63% (52-73%), stunting 24% (15-34%) and neuro-developmental delay 17% (8-25%). Delay in one aspect of neuro-development was seen in 54% (43-66%). Early diagnosis was protective against poor control of disease (adjusted Odds Ratio, ORa=0.24 [CI: 0.08-0.77]). Trends towards protection were seen for initial and continuing specialist care. For delay in at least one cognetive aspect, early diagnosis was found to be protective (ORa=0.19 [CI 0.05-0.76]); results for specialist care were inconclusive. For stunting, low parent education was found to be a risk factor. (ORa of 5.45 [CI: 1.3-22.7]). Conclusion. Fifty-four percent of the study patients had delay in one aspect of neuro-development. While other factors play a role in the outcome of CH, early diagnosis and treatment were shown to be protective of patients from poor control of disease and cognitive delays. Observed trends of positive benefits of specialist care at onset and continuing medical management, and the association of low parent education with poor growth should be considered in drafting specific guidelines for the long term follow-up care and monitoring of CH patients detected through newborn screening. The low percentage of participation and incomplete retrieval of information are major limitations of this retrospective study. This stresses the need for better monitoring tools that will ensure proper tracking, medical care and evaluation of CH patients.

Cost-benefit analysis of the newborn screening program of the Philippines

BACKGROUND: Newborn Screening (NBS) is a public health activity aimed at the early identification of infants who are affected by certain genetic/metabolic/infectious conditions. A cost analysis is critical for national implementation for integration as a public health program. OBJECTIVES: 1) To determine the incidence rates of congenital hypothyroidism (CH), congenital adrenal hyperplasia (CAH), galactosemia (GAL), phenylketonuria (PKU) and glucose-6-phosphate dehydrogenase (G6PD) deficiency; and 2) To determine whether NBS is cost-beneficial for each disorder individually or in combination, from a societal perspective. STUDY DESIGN: Cross sectional survey and cost-benefit analysis. SUBJECTS AND METHODS: The study was conducted through a screening survey of the original 24 Metro Manila hospitals. Newborns were screened for CH, CAH, GAL, PKU and G6PD deficiency after the 24th hour of life. Those who screened positive underwent serum confirmatory testing. Using incidence rates from the screening survey, a population of 1.5 million, and different screening combinations, the costs for the detection and treatment of the five disorders were compared to the benefits projected from preventing the corresponding complications and consequent productivity losses. For economic evaluation, we compared sequential analysis of doing tandem/multiple testing for the different disorders vs a "do-nothing" alternative. Sensitivity analyses for different incidence and discount rates were conducted to test the strength of the conclusions. RESULTS: The incidences of the disorders with 95% confidence intervals are: CH is 1:3 235 (1:2 219 - 1:5 946); CAH is 1:7 455 (1:4 046 - 1: 14245); GAL is 1: 106 006 (1: 44 218-1:266 796); and G6PD deficiency is 1:167 (1:151 - 1: 186). Screened individually, CH and G6PD deficiency had net benefits of US$ 5.29 M and US$ 15.44 M, respectively. The other conditions yielded net costs when screened individually - CAH (US$ 2.61 M), GAL (US$ 0.90 M) and PKU (US$ 6.74 M). Pairing the disorders with CH showed the following benefit:cost ratios - CH + CAH, 1.3; CH + GAL, 2.0; CH + G6PD deficiency, 3.4; and CH + PKU, 0.9. Combining disorders resulted in the following benefit:cost ratios - CH + CAH + GAL, 1.2; CH + CAH + GAL + PKU, 0.8; and CH + CAH + GAL + G6PD deficiency, 2.1. Screening for the 5 disorders in tandem resulted in a benefit:cost ratio of 1.4 and a net benefit of US$ 11.42 M.

Mutations of the phenylalanine hydroxylase (PAH) gene in Filipino patients with phenylketonuria

INTRODUCTION: Phenylketonuria (PKU), an autosomal recessive metabolic disorder caused by phenylalanine hydroxylase (PAH) deficiency, leads to hyperphenylalaninemia and neurological damage if untreated. This is the first study in the Philippines to identify the disease-causing mutations in the PAH gene of clinically diagnosed Filipino PKU patients. METHODS: The study included four unrelated PKU patients detected by the Philippine Newborn Screening Program from 1996 to 2008. Plasma amino acid analyses for all patients showed increased phenylalanine and low to normal tyrosine levels consistent with the diagnosis of PKU. Mutations in the PAH gene were identified by genomic DNA extraction from dried blood spots of the patients, PAH exon amplification by polymerase chain reaction and subsequent bi-directional DNA sequence analysis. RESULTS: All patients presented with significantly elevated phenylalanine levels on bacterial inhibition assay and thin layer chromatography. Urinary pterins confirmed the diagnosis of Tetrahydrobiopterin deficiency in two patients while the other 2 patients had the Classical PKU phenotype. Four previously identified mutations in the PAH gene (p.I65T, p.R413P, p.EX6-96A>G, p.R243Q) were identified in those with Classical PKU. CONCLUSION: The present results confirm the heterogeneity of mutations at the PAH locus in Filipinos. Neonatal screening and the use of molecular diagnosis significantly aid in the medical management and genetic counseling of patients and their families.

Mutations of the steroid 21-hydroxylase gene among Filipino patients with congenital adrenal hyperplasia

Congenital adrenal hyperplasia (CAH), an autosomal recessive disorder, is due to deficiency of the enzymes involved in adrenal steroidogenesis. Phenotypic manifestations vary as a result of the degree of glucocorticoid or mineralocorticoid deficiency and androgen excess present. Among Filipinos, the estimated crude incidence of CAH is approximately 1 in 7,000, which is higher than what is reported in most populations. More than 90% of all cases result from a 21-hydroxylase (21-OH) (cytochrome P450c21) enzyme deficiency involving two 21-OH genes, the active gene (CYP21) and a pseudogene (CYP21P). Studies have shown that mutations result from unequal crossover during meiosis which leads to complete deletion of the gene, gene conversion events or to point mutations. To date, there are no published data on the types of mutations present among Filipinos diagnosed with congenital adrenal hyperplasia. The objective of this study is to describe the profile of Filipino patients diagnosed with CAH and to determine the disease-causing alleles in the 21-OH gene of these patients. Using a method of combined differential polymerase chain reaction and amplification created restriction site approach, direct probing for the presence of known mutations in exons 1,3,4,6,7,8 and intron 2 of the CYP21 and CYP21P genes among Filipino patients with CAH was performed. A total of 12 unrelated CAH patients were examined. A majority of these cases had a premature splicing error mutation at nucleotide 656 of intron 2. The determination of the most frequent alleles in our population can facilitate rapid screening for mutations in the 21-OH gene and lead to a definitive diagnosis of CAH.

Improved screening efficiency for phenylketonuria using a modified bacterial inhibition assay protocol- Autoclaving the bloodspot

The Guthrie bacterial inhibition assay (BIA) tests for elevated phenylalanine (PHE) by measuring B. subtilis growth zone density in an agar medium. Dried blood spots with elevated PHE on initial BIA screening undergo repeat BIA testing and thin-layer chromatography (TLC). Specimens with elevated PHE by TLC or BIA on second-tier testing require recall. To streamline PKU screening and reduce the recall rate, we tested a modified BIA protocol incorporating autoclaving of dried blood spots. Autoclaving improves growth zone appearance and has been previously reported to reduce the number of specimen requiring repeat testing. From June to October 2006, dried blood spot samples with initially elevated PHE were autoclaved at 110°C for 5 min, then retested by BIA. Samples with still-elevated PHE were analyzed by TLC. 1078 of 37,268 samples (2.89%) had initially elevated PHE. After autoclaving, 1036 no longer exhibited elevated PHE decreasing to 42 (0.11%) the number requiring TLC. By comparison, the unmodified algorithm resulted in 3.14% of samples received from July - December 2006 requiring both repeat BIA and TLC testing. We have since modified our PKU screening algorithm to require repeat BIA testing from autoclaved samples prior to TLC analysis. This translates to a significant reduction in time and resources for second-tier testing and follow-up, and prevents stress for the parents of a newborn who would have been recalled unnecessarily.

Detection of maple syrup urine disease on newborn screening second tier testing for phenylketonuria

Newborn screening for phenylketonuria (PKU) in the Philippines uses a bacterial inhibition assay to detect elevations in phenylalanine (PHE). The BIA sensitivity is affected by substances such as antimicrobials. Semiquantitative second-tier screening with thin layer chromatography (TLC) verifies inconclusive PKU screens. Maple syrup urine disease (MSUD) is a prevalent inborn error of metabolism in the Philippines that is currently not part of the Philippine newborn screening program. We report on the incidental detection of MSUD by second-tier TLC PKU screening in order to begin to establish the evidence necessary for its inclusion in the Philippine Newborn Screening Program. We reviewed the PKU newborn screening database from September 1, 1996 to June 12, 2008 to document the number of cases of elevated LEU detected incidental to confirming PHE elevations by second-tier TLC. Elevated LEU findings were studied further to document the number of MSUD cases. From September 1, 1996 to June 12, 2008, 966,096 babies were screened for PKU and 28,248 (2.9%) required second-tier testing. Of these, 403 had elevated PHE and 9 were confirmed to have either classic PKU or hyperphenylalaninemia. Fifty-three of 28,248 babies had normal PHE concentrations but elevated LEU concentrations. These babies were recalled and a second dried blood spot was requested. Of these, 15 had elevated LEU and were subsequently confirmed to have MSUD. Two babies had concurrent elevations of PHE and LEU, but both were deceased at the time of recall. Confirmation of 15 MSUD cases was almost twice as high as the number of PKU/HPA confirmed cases. Since MSUD patients were detected incidental to PKU screening and there was no initial MSUD screening, the incidence of MSUD is almost certainly much greater. The number of MSUD cases incidentally detected confirms that MSUD exists at a significantly higher prevalence in the Philippine newborn population than PKU, and its inclusion in the newborn screening panel should be considered as soon as feasible.

Galactosemia in three Filipino patients - The importance of newborn screening

Disorders of galactose metabolism can be fatal if not treated early. Newborn screening has made it possible to detect and treat this disease. Three cases of galactosemia, one with galactokinase deficiency and two with galactose-1-phosphate uridyltransferase deficiency detected by newborn screening, are presented. Because of early detection and management, the first patient was spared the early complications of galactosemia and continues to grow and develop normally. The two other patients were diagnosed at 1 month, initial presentation included hepatomegaly and failure to thrive. Institution of treatment was able to reverse the acute complications and both are currently doing well. The importance of galactosemia newborn screening in preventing complications resulting from the disease is emphasized.

Initial versus confirmatory thyroid stimulating hormone (TSH) levels: is there a correlation?

Newborn screening for congenital hypothyroidism (CH) in the Philippines began in 1996. The screening method used is the fluoroimmunometric assay of thyroid stimulating hormone (TSH) from dried blood spot. In the past five years (June 1996--Sept 2001), 176,548 newborns have been screened. Of these, 237 had elevated TSH levels and 51 (22%) were confirmed to have CH. One hundred forty-six (61%) had normal TSH levels on confirmatory testing; five (2%) expired; 25 (11%) were lost to follow-up, while 10 (4%) were being recalled at the time of this study. Thirty-three out of 51(65%) CH patients are female. Only 38 of 51 patient charts were available for data analysis. Thirteen of 51 CH patients were lost to follow-up after confirmation of the disorder. The mean age at which levo-thyroxine was initiated is 1 1/2 months at a modal dosage of 25 microg OD. The initial TSH levels as determined by the Philippine Newborn Screening Laboratory directly correlates with the confirmatory TSH levels done in other endocrine laboratories (Spearman's rho=0.57, p value=0.0002, at a=0.05). However, the time of heel prick on the newborn was independent of the TSH levels, (Spearman's rho=-0.16, p value=0.377 at a=0.05) hence there was no significant difference with respect to the initial TSH level of blood samples taken at 48 hours, less than one week, one to two weeks; or even more than two weeks after birth (Kruskall Wallis test, p value=0.064 at a=0.05). Using Fisher's exact test, there is no sufficient evidence to say that there is an association between gender and the incidence of CH among screened newborns whose TSH levels were initially elevated (p 2-tailed=0.183, p 1-tailed=0.113 at a=0.05).

Hyperphenylalaninemia in the Philippines.

To present patients with hyperphenylalaninemia (HPA) diagnosed by routine newborn screening and to discuss the principles in managing hyperphenylalaninemia, retrospective clinical chart review was conducted. Newborn screening for phenylketonuria (PKU) was performed using the Guthrie Test or Bacterial Inhibition Assay, utilizing dried blood spots on special filter cards. Positive screens were confirmed through plasma amino acid determination, urinary pterins for tetrahydrobiopterin deficiency, enzyme analysis. Once confirmed, the patients were kept on low-phenylalanine diet and regularly monitored for blood phenylalanine levels and developmental profile. A total of 189,720 newborns were screened from 1996--2001. Seventy five screened positive for PKU; 41 returned for retest; 3 were confirmed positive for HPA. This paper presents the first two cases of HPA detected by the Philippine Newborn Screening Program. The management of each case upon diagnosis is discussed. The significance of early detection and treatment of HPA is emphasized.

6-pyruvoyl tetrahydropterin synthase deficiency: a case report.

A 5 day old girl screened positive for hyperphenylalaninemia on routine newborn screening. Initial diagnostic work-up showed elevated blood phenylalanine of 1100 mmol/L and low tyrosine. Limited protein diet and phenylalanine-free formula were prescribed. Further investigation revealed a defect in biopterin metabolism. Urine had no detectable biopterin (BH4) and an elevated level of neopterin at 24.31 mmol/mole Cr. Enzymatic assay showed zero level of 6-pyruvoyl tetrahydropterin synthase. The activity in the mother was 3.5 or 19.9% of controls consistent with heterozygosity. The concentrations of 5-hydroxyindoleacetic acid and homovanillic acid in the cerebrospinal fluid were below the reference ranges. A treatment regimen of BH4 tablets, 5 hydroxytryptophan and DOPA was initiated. The diagnostic evaluation, management and follow-up of patients with this disorder will be outlined. This is the first reported case of a Filipino with a defect in biopterin metabolism.