Phenylketonuria—The Guthrie Screening Test—A Method of Quantitation,Observations on Reliability and Suggestions for Improvement

The Guthrie bacterial inhibition assay method of screening neonatal infants for phenylketonuria (pku) initiated mass screening for inborn errors of metabolism. It is a simple, cheap procedure admirably suited to private local use as against private or public central use. However, using parallel fluorometric determinations as a basis for comparison, and 4 mg per 100 ml serum phenylalanine as a presumptive positive threshhold, the Guthrie test yielded 53 per cent “false negatives.” Extrapolating from a combination of our data and reported phenylalanine levels at three days of age or less in proved pku patients, it is estimated the Guthrie test might fail to detect one of 25 pku patients screened at three days of age or less. Means to diminish this risk are considered.     

Case Finding in Phenylketonuria: II. The Guthrie Test

Experience with over 6000 Guthrie tests is presented. This test is a screening procedure for phenylketonuria using small amounts of blood spotted on a filter paper which are tested by a bacterial “inhibition assay”. Certain technical aspects of the test (e.g. relation between the concentration of phenylalanine in the blood and extent of the bacterial growth zones produced, type of filter paper, size of the blood spot on the paper) were investigated. It was shown that the Guthrie test clearly distinguishes between subjects with normal plasma phenylalanine levels and patients with untreated phenylketonuria.Applications of the Guthrie test in screening a mental hospital population, admissions to a penitentiary and newborn babies are described.     

A quantitative bacterial micro-assay for rapid detection of serum phenylalanine in dry blood-spots: Application in phenylketonuria screening

Phenylketonuria is an inherited metabolic disease, which is characterized by increased level of serum phenylalanine (Phe). The quantitative measurement of Phe in the serum is necessary to confirm the disease, and to distinguish phenylketonuria from other forms of hyperphenylalaninemia. In this study, we report a rapid and inexpensive micro-assay for simultaneous detection and quantitative measurement of serum Phe in dry blood-spots. Analysis of the standard curve showed a broad linear Phe range of 120-1800 micromol L(-1). Application of this method in conjunction with the standard Guthrie bacterial inhibition assay and high-pressure liquid chromatography in analyzing 34 samples from phenylketonuria patients and control samples produced comparable results, with the regression equation of Y= 0.994X + 0.996. The advantage of this method over the Guthrie bacterial inhibition assay is its ability to measure the serum Phe quantitatively without false positive results. The method was successfully applied to dried blood-spots as well as serum and whole blood samples. The cost per sample is about 20-50 US cents, which is much less than those of high-pressure liquid chromatography and enzymatic commercial kits. The method can be automated, which is suitable for neonatal and mass phenylketonuria screening, especially in developing countries, where funding is a limiting factor.     

Fifteen-year experience with screening for phenylketonuria with an automated fluorometric method.

An automated fluorometric method, rather than the Guthrie test, has been used in North Carolina for neonatal screening for phenylketonuria (PKU). Although there is no testing law, 97% of newborn infants are screened. Twelve children with PkU, not referred for dietary management, were born before the screening program was established, were born elsewhere, or were successfully identified at birth but not referred for treatment. None was missed because of laboratory error or because of the lack of a testing law. Positive skewing was noted among initial blood phenylalanine levels of 49 infants with PKU and severe hyperphenylalaninemia. Log transformations caused the values to be normally distributed and permitted the calculation of tolerance and confidence limits. These provided estimates of the percentage of phenylketonuric infants whose initial blood levels might fall below any given cutoff value.     

Molecular genetics of phenylketonuria in Mediterranean countries: a mutation associated with partial phenylalanine hydroxylase deficiency.

We report the characterization of a mutation in the phenylalanine hydroxylase (PAH) gene associated with partial residual activity of the enzyme. This point mutation (280glu----lys) was found by sequencing a mutant cDNA clone derived from a needle biopsy of the liver in a child with variant form of phenylketonuria. There is a strict concordance between homozygosity for the mutation and this particular phenotype. The (280glu----lys) mutation is linked to an original and rare RFLP haplotype at the PAH locus found in south Europe and North Africa. So far, this genotype-haplotype association is both inclusive and exclusive. Thirty-three PAH-deficient patients were screened for the mutation by using polymerase chain-reaction amplification of their genomic DNA extracted from Guthrie cards. Since a large number of patients can be screened for a particular mutation by using Guthrie cards, the possibility arises of using these samples collected by national newborn screening centers for prospective and retrospective detection of other mutations in the human genome.     

Prevalence of HIV antibody and pregnancy in Tayside, 1984-9: background to screening.

OBJECTIVE--To determine age specific prevalence of HIV antibody, incidence of pregnancy, and likelihood of detection and correct assignment to risk category by antenatal screening of women known to be positive for HIV antibody, from 1984 to 1989. DESIGN--Retrospective analysis of reproductive history and risk behaviour of women positive for HIV antibody and prediction of detection by screening on the basis of blood group samples, Guthrie tests, and rubella tests. SETTING--City of Dundee, where the prevalence of HIV is high, since the appearance of HIV in 1984, predominantly among heterosexual intravenous drug users. PATIENTS--All (61) women known to be positive for HIV antibody who had had clinically indicated tests, for whom case notes were available for 60. MAIN OUTCOME MEASURES--Risk assessment according to case notes and reported to the laboratory, incidence of infection, geographical location, age, date of positive test result, and reproductive history. RESULTS--With 61 infected women the overall minimum prevalence among women within the city of Dundee was 0.67/1000 and 2.9/1000 among women in their third decade. Of the 60 women whose reproductive history was available, 35 had 57 pregnancies, 36 of which occurred after seroconversion was known to have taken place, representing 8.7% of the total number of affected pregnancies reported for the United Kingdom. If antenatal screening for HIV had been performed between 1984 and 1989 it could not have detected positivity for HIV antibody in 25 (42%) women who had no pregnancies during this time. Among the remaining 35 women, screening samples taken for blood grouping could have identified a maximum of 34 (57%), samples taken to check rubella susceptibility a maximum of 22 (37%), and blood spots on Guthrie cards a maximum of 19 (32%). Retesting would have occurred in 14 women 33 times with samples taken for blood grouping, but three and four women would have been tested twice using samples taken for rubella testing and Guthrie cards respectively. Anonymous screening would have been unable to determine risk category as a history of intravenous drug use was known in 47 (79%) women before testing but this was increased by a further 5 (8%) who admitted to it after the test result was known. CONCLUSION--Interpreting the results of antenatal screening programmes will be complex and will underestimate overall prevalence of HIV antibody among women; this will be exaggerated by strategies based on anonymous testing with Guthrie cards or on samples taken for rubella testing, which do not include women who have had an earlier loss of pregnancy. Only open testing with consent will permit satisfactory attribution to     

Neonatal detection of the 35delG mutation of the GJB2 gene in families at risk for deafness

About half of congenitally deaf children that have a recessively inherited sensorineural deafness are born from normal-hearing parents and have no risk factor for hearing loss. Mutation 35delG in the connexin-26 gene is in European populations the basis for around half of all recessively inherited prelingual sensorineural deafness. The aim of our study was to assess the efficacy and utility of the 35delG mutation of the connexin-26 gene analysis for neonates at familial risk, from DNA isolated from Guthrie newborn screening cards. Newborns who had consanguineous parent and/or a familial history of deafness underwent connexin-26 gene analysis from DNA isolated from Guthrie cards and two hearing screening tests (transient evoked otoacoustic emissions, and auditory brainstem recordings). 24 newborns were includes in this pilot study; one of them is homozygous for the 35delG mutation and had abnormal hearing screening tests; all the others newborns had normal connexin gene and at least one normal hearing screening test. Detection on connexin-26 gene mutation is feasible in selected at-risk newborns on one additional blood spot on Guthrie card.     

Connexin-26 gene analysis in hearing-impaired newborns

The efficacy and utility of the Connexin-26 (Cx-26) gene (also called GJB2) analysis from DNA isolated from Guthrie newborn screening cards is demonstrated. This analysis precisely defined a major cause of prelingual nonsyndromic deafness in those children requiring amplification in our study. Guthrie cards were obtained from 49 deaf children requiring amplification identified over the last 5 years by the Rhode Island Newborn Screening Program. Children with syndromes or other recognizable causes of hearing loss were excluded. DNA was extracted from the Guthrie cards and analyzed sequentially for the Cx-26 35delG mutation and then for the 167delT mutation followed by gene sequencing on remaining heterozygotes. Three of 42 children were 35delG homozygotes; 2/42 children were 35delG/167delT compound heterozygotes. One child was identified as being a 35delG heterozygote with no other mutation found by sequencing. Nine Guthrie cards yielded no amplification or uninterpretable results. Cx-26 mutations were identified as causing 11.9% of the deafness in the children studied. In conclusion, Cx-26 analysis is an important test that identifies a major cause of prelingual nonsyndromic deafness. Molecular analysis of hearing-impaired newborns will be important for genetic counseling in these families. Failures with Guthrie cards may make use of other collection methods preferable.     

Placental transfer of maternally-derived IgA precludes the use of guthrie card eluates as a screening tool for primary immunodeficiency diseases

There is a need for neonatal screening tools to improve the long-term clinical outcome of patients with primary immunodeficiency diseases (PID). Recently, a PCR-based screening method for both TRECs and KRECs using Guthrie card samples has been developed. However, the applicability of these excision circle assays is limited to patients with severe T or B cell lymphopenia (SCID, XLA and A-T), whereas the most common forms of PID are not detected. Absence of serum IgA is seen in a major fraction of patients with immunological defects. As serum IgA in newborns is considered to be of fetal origin, eluates from routinely collected dried blood spot samples might thus be suitable for identification of children with PID. To assess the applicability of such screening assays, stored Guthrie card samples were obtained from 47 patients with various forms of primary immunodeficiency diseases (SCID, XLA, A-T, HIGM and IgAD), 20 individuals with normal serum IgA levels born to IgA-deficient mothers and 51 matched healthy newborns. Surprisingly, normal serum IgA levels were found in all SCID, XLA, A-T and HIGM patients and, additionally, in all those IgAD patients born to IgA-sufficient mothers. Conversely, no serum IgA was found in any of the 16 IgAD patients born by IgA-deficient mothers. Moreover, half of the IgA-sufficient individuals born by IgA-deficient mothers also lacked IgA at birth whereas no IgA-deficient individuals were found among the controls. IgA in neonatal dried blood samples thus appears to be of both maternal and fetal origin and precludes its use as a reliable marker for neonatal screening of primary immunodeficiency diseases.     

Intellectual level (IQ) in heterozygotes for phenylketonuria (PKU)

Summary There is a statistically significant difference in the IQ's of PKU and histidinemia parents. The difference is due entirely to the verbal part of the Hamburg-Wechsler test. There is no significant difference in performance. The heterozygous state of histidinemia does not seem to bear an intellectual (evolutionary) advantage, since the IQ's of histidinemia parents show the same distribution as a normal population. In early and mostly well-treated PKU patients, the same slight deficit in verbal IQ appears with increasing age (changing test methods). These patients, simultaneously tested at 4 years of age with the Bühler-Hetzer and Kramer tests, exhibit a statistically significant difference between the results in favor of the less verbal Bühler-Hetzer. Since heterozygots, for PKU never have elevated phenylalanine blood levels, and because tryosine deficiency as argued by others seems highly improbable, we believe that the PKU gene has a more direct action on (or in) at least certain ganglion cells, lowering the verbal IQ slightly, but significantly. This action is not reflected by phenylalanine increase in the extracellular space in heterozygots and is not abolished by dietary treatment in homozygous PKU patients. The major damage in PKU patients must be due to chronic phenylalanine poisoning, which deteriorates cells and/or functions on a much larger scale, because it can be easily prevented by decreasing the phenylalanine blood level with correct dietary treatment.     

Screening of newborn infants for cholestatic hepatobiliary disease with tandem mass spectrometry

ObjectiveTo assess the feasibility of screening for cholestatic hepatobiliary disease and extrahepatic biliary atresia by using tandem mass spectrometry to measure conjugated bile acids in dried blood spots obtained from newborn infants at 7-10 days of age for the Guthrie test.SettingThree tertiary referral clinics and regional neonatal screening laboratories.DesignUnused blood spots from the Guthrie test were retrieved for infants presenting with cholestatic hepatobiliary disease and from the two cards stored on either side of each card from an index child. Concentrations of conjugated bile acids measured by tandem mass spectrometry in the two groups were compared.Results218 children with cholestatic hepatobiliary disease were eligible for inclusion in the study. Two children without a final diagnosis and five who presented at <14 days of age were excluded. Usable blood spots were obtained from 177 index children and 708 comparison children. Mean concentrations of all four bile acid species were significantly raised in children with cholestatic hepatobiliary disease and extrahepatic biliary atresia compared with the unaffected children (P<0.0001). Of 177 children with cholestatic hepatobiliary disease, 104 (59%) had a total bile acid concentration >33 μmol/l (97.5th centile value for comparison group). Of the 61 with extrahepatic biliary atresia, 47 (77%) had total bile acid concentrations >33 μmol/l. Taurotrihydroxycholanoate and total bile acid concentrations were the best predictors of both conditions. For all cholestatic hepatobiliary disease, a cut off level of total bile acid concentration of 30 μmol/l gave a sensitivity of 62% and a specificity of 96%, while the corresponding values for extrahepatic biliary atresia were 79% and 96%.ConclusionMost children who present with extrahepatic biliary atresia and other forms of cholestatic hepatobiliary disease have significantly raised concentrations of conjugated bile acids as measured by tandem mass spectrometry at the time when samples are taken for the Guthrie test. Unfortunately the separation between the concentrations in these infants and those in the general population is not sufficient to make mass screening for cholestatic hepatobiliary disease a feasible option with this method alone.

Key messages

• The prognosis of cholestatic hepatobiliary disease in infancy, in particular biliary atresia, is improved by early detection
• Infants destined to present with cholestatic jaundice in the first few months of life have raised concentrations of bile acids in the blood spots obtained at 7-10 days for current neonatal screening programmes
• Tandem mass spectrometry can be used to detect this marker of neonatal cholestasis
• Unfortunately there is too much overlap between bile acid concentrations in infants with cholestasis and those in control infants for this to be used as a single screening test for cholestatic hepatobiliary disease in general and biliary atresia
• Tandem mass spectrometry is a powerful tool for neonatal screening but every potential application must be carefully assessed

     

Testing for the chemical induction of aneuploidy in the male mouse

Dyad scores of metaphase II spermatocytes in the mouse have been used as an end point to assess the aneuploidy-inducing potential of three different chemicals; p-fluorophyalanine, phenylalanine and 6-mercaptopurine. The sensitivities of three different spermatogenic stages have been tested; pre-leptotene, zygotene and metaphase I. No effect was found at any treated stage for 6-mercaptopurine and phenylalanine. p-Fluorophenylalanine, when compared to control treatments, did, however, induce non-disjunction when applied at metaphase I. It also caused a delay to spermatogenesis when applied at this stage. The potential of mammalian test systems for the routine screening of chemicals as non-disjunction inducers, is discussed.     

Phenylketonuria variants in Ontario.

Since mass screening of the newborn population for phenylketonuria (PKU) by the Guthrie test was begun in Ontario in July 1965 many variants of PKU have been recognized in the 96 to 97% screened. Seventy-one cases of classic PKU were detected (four were missed). Of 48 cases of persistent hyperphenylalaninemia discovered, 18 were classified as atypical PKU and 30 as persistent benign hyperphenylalaninemia. Numerous infants with transient hyperphenylalaninemia (initial values over 10 mg/dl in 12), in many instances the result of transient neonatal tyrosinemia, were discovered. There was a slight predominance of males. Serum phenylalanine values of up to 15 mg/dl seemed to be harmless to the developing brain. A survey of 67 247 adults in the general population revealed 1 person with PKU and 1 with persistent benign hyperphenylalaninemia; both had normal intelligence quotients. Of 1548 mothers of retarded children tested, none had hyperphenylalaninemia.     

Investigations of antithrombin III-activity in patients after myocardial infarction and a long-term coumarin therapy

In 40 M.I. patients with long-term anticoagulant treatment, (Falithrom), the AT III-activity was determined at an interval of three years by means of Chromozym TH. We have found a mean AT III-value in the first testing period of 79.67 (+/- 14.16) per cent and in the second assessment of 82.5 (+/- 10.42) per cent. The difference is not significant. However, we were unable to confirm the comparatively marked increase AT III for dicoumarol treatment found by Roka and Bleyl. In acute M.I.-patients was demonstrated a decreased AT III-activity in the first 3-4 days and a normalisation tendency in the next 10-14 days. Our values of the mean AT III-activity were in the lower normal range for patients with long-term coumarin therapy. In dead patients (average age 70 years) there is a trend of a risk to an untimely death in the presence of pathologic AT III-activity (despite a good anticoagulation of an individual mean quick test from greater than or equal to 0.20 to less than or equal to 0.30) or a bad anticoagulation (mean individual quick tests greater than or equal to 0.30 to 0.35), but a normal AT III-activity. The three dimensional analysis was not significant.     

Blood-Based Protein Biomarker Panel for the Detection of Colorectal Cancer

Background

The majority of colorectal cancer (CRC) cases are preventable by early detection and removal of precancerous polyps. Even though CRC is the second most common internal cancer in Australia, only 30 per cent of the population considered to have risk factors participate in stool-based test screening programs. Evidence indicates a robust, blood-based, diagnostic assay would increase screening compliance. A number of potential diagnostic blood-based protein biomarkers for CRC have been reported, but all lack sensitivity or specificity for use as a stand-alone diagnostic. The aim of this study was to identify and validate a panel of protein-based biomarkers in independent cohorts that could be translated to a reliable, non-invasive blood-based screening test.

Principal Findings

In two independent cohorts (n = 145 and n = 197), we evaluated seven single biomarkers in serum of CRC patients and age/gender matched controls that showed a significant difference between controls and CRC, but individually lack the sensitivity for diagnostic application. Using logistic regression strategies, we identified a panel of three biomarkers that discriminated between controls and CRC with 73% sensitivity at 95% specificity, when applied to either of the two cohorts. This panel comprised of Insulin like growth factor binding protein 2 (IGFBP2), Dickkopf-3 (DKK3), and Pyruvate kinase M2(PKM2).

Conclusions

Due to the heterogeneous nature of CRC, a single biomarker is unlikely to have sufficient sensitivity or specificity for use as a stand-alone diagnostic screening test and a panel of markers may be more effective. We have identified a 3 biomarker panel that has higher sensitivity and specificity for early stage (Stage I and -II) disease than the faecal occult blood test, raising the possibility for its use as a non-invasive blood diagnostic or screening test.     

Predictors of chlamydial infection and gonorrhea among patients seen by private practitioners.

OBJECTIVE: To identify the predictors of chlamydial infection and gonorrhea among patients tested by general practitioners. DESIGN: Prospective study. SETTING: General private practice, family planning and abortion clinic, adolescent clinic, sexually transmitted disease (STD) clinic and community health clinic in downtown Montreal. PATIENTS: The 2856 patients were included because of symptoms compatible with an STD, a history of sexual contact with a person known or suspected to have chlamydial infection, a history of a nonexclusive sexual relationship or presentation for an abortion. MEASURES: Patient information was obtained by the attending physician on a standard form. Enzyme immunoassay (EIA) for Chlamydia trachomatis and culture for Neisseria gonorrhoeae were performed on cervical (female) or urethral (male) samples. Stepwise logistic regression was used to identify the predictors of infection. RESULTS: The EIA results were positive in 11.1% of the cases and the culture results in 2.3%. Among the males chlamydial infection was independently associated with low age (odds ratio [OR] = 0.88 per year), heterosexuality (OR = 4.99), urethral discharge (OR = 3.74) and the absence of a history of gonorrhea (OR = 0.51). Gonorrhea was associated with urethral discharge (OR = 24.3) and homosexuality (OR = 3.68). Among the females chlamydial infection was associated with low age (OR = 0.79 per year), a history of sexual contact with a person known to have chlamydial infection (OR = 2.30), multiple sexual partners in the previous 12 months (OR = 1.60) and a reason for the test other than screening purposes (OR = 0.60). Gonorrhea was associated with a reason other than screening (OR = 0.24) and low age (OR = 0.74 per year). Among the patients tested for screening purposes age was the only significant predictor of chlamydial infection (OR = 0.79 per year), and the prevalence of gonorrhea was 0.4%. The actual rate of chlamydial infection was 11.8% among the patients younger than 25 years, 5.7% among those 25 to 34 years and 0.6% among those over 34. CONCLUSIONS: Age alone can be used as a criterion to screen for chlamydial infection among asymptomatic patients without a history of sexual contact with a person known or suspected to have such infection and with a history of a nonexclusive relationship. The prevalence in our population justifies screening people up to 34 years of age.     

The promise and challenges of blood spot methylomics

Epigenome-wide association studies (EWAS) are being extensively performed to identify epigenetic variants associated to complex diseases. However, EWAS may identify variants that are disease-induced rather than disease-causal. Recent studies have highlighted the use of Guthrie cards to profile the methylome at birth, permitting researchers to find epigenetic variants present in patients before they are diagnosed with clinical disease, with the implicit suggestion that these variants are more likely to be disease causal. The use of Guthrie cards for research purposes throws up a number of ethical issues. We review here the promises and pitfalls of Guthrie cards for disease research.     

Screening for squamous cervical cancer: duration of low risk after negative results of cervical cytology and its implication for screening policies. IARC Working Group on evaluation of cervical cancer screening programmes.

A collaborative study of screening programmes in eight countries was performed to estimate the risks of cervical cancer associated with different screening policies. Most of the data came from centrally organised screening programmes. Relative protection was higher in women who had had two or more negative results of screening tests than in those who had had only one negative smear, particularly in the first five years after the last test. There was little difference in the protection afforded by screening every year compared with every three years, but screening only once every five or 10 years offered appreciably less protection. The age of the women did not affect the sensitivity of the test or the sojourn time of the disease (the length of the detectable preclinical phase during which abnormal cytology could be picked up if a smear were taken); invasive cancer in women under 25 was rare. Centrally organised screening programmes were more effective than uncoordinated screening. Screening programmes should be aimed principally at women aged 35-60 but should start some years before the age of 35, and the intervals between screening should be three years or less.     

Comparison of Methods for Assessing Resistance to Meloidogyne arenaria in Peanut

Use of resistant cultivars is a desirable approach to manage the peanut root-knot nematode (Meloidogyne arenaria). To incorporate resistance into commercially acceptable cultivars requires reliable, efficient screening methods. To optimize the resistance screening protocol, a series of greenhouse tests were done using seven genotypes with three levels of resistance to M. arenaria. The three resistance levels could be separated based on gall indices as early as two weeks after inoculation (WAI) using 8,000 eggs of M. arenaria per plant, while four or more weeks were needed when 1,000–6,000 eggs/plant were used. High inoculum densities (over 8,000 eggs/plant) were needed to separate the three resistance levels based on eggs per gram of root within eight WAI. A gall index based on percentage of galled roots could separate the three resistance levels at lower inoculum levels and earlier harvest dates than other assessment methods. The use of eggs vs. second-stage juveniles (J2) as inoculum provided similar results; however, it took three to five more days to collect J2 than to collect eggs from roots. Plant age affected gall index and nematode reproduction on peanut, especially on the susceptible genotypes AT201 and D098. The genotypes were separated into their correct resistance classes when inoculated 10 to 30 days after planting, but were not separated correctly when inoculated on day 40.     

Appraisal of a new scheme for prenatal screening for Down's syndrome.

OBJECTIVE--To appraise a new method of prenatal screening for Down''s syndrome based on maternal serum concentrations of alpha fetoprotein, unconjugated oestriol, and human chorionic gonadotrophin combined with maternal age--the "triple test." DESIGN--Examination of the cost effectiveness of the triple test relative to screening only by maternal age over a range of population detection rates. SETTING--Leicestershire Health Authority. MAIN OUTCOME MEASURES--Costs per affected fetus detected. RESULTS--The triple test is more cost effective than screening only by maternal age for risk cut off points for amniocentesis, resulting in a detection rate over 45%. The most efficient detection rate is around 60-65%, for which the cost per case detected is around 29,000 pounds, through screening with higher detection rates is still likely to be cost beneficial. CONCLUSIONS--Prenatal screening for Down''s syndrome based on the triple test should replace screening based only on maternal age. Individual women''s preferences should be elicited by the use of structured decision analysis in order to maximise utility and so increase the benefits of the screening programme.