Neil A. Holtzman

Phenylketonuria Due to a Deficiency of Dihydropteridine Reductase

The onset of neurologic symptoms in a child who had markedly elevated blood phenylalanine levels during the first two weeks of life and who was promptly treated with a low phenylalanine diet, with excellent control of serum phenylalanine levels, suggested that this child had an unusual form of phenylketonuria. In assays of the components of the phenylalanine hydroxylating system (open liver biopsy at 14 months), the activity of phenylalanine hydroxylase was 20 per cent of the average normal adult value. By contrast, no dihydropteridine reductase activity was detected in the patients liver, brain or cultured skin fibroblasts. Since dihydropteridine reductase is also essential for the biosynthesis of dopamine, norepinephrine, and serotonin, disturbed neurotransmitter function may be responsible for the patients neurologic deterioration. On the basis of these results, assay of reductase in cultured skin fibroblasts may be advisable in the initial diagnosis of phenylketonuria.

Effect of Age at Loss of Dietary Control on Intellectual Performance and Behavior of Children with Phenylketonuria

We determined the effect on intellectual performance and behavior of the age at which dietary control was lost in 119 10-year-old children with phenylketonuria (PKU) who had started on a diet low in phenylalanine before the age of 65 days. The childrens diets were considered to be out of control when their blood phenylalanine concentration persistently exceeded 15 mg per deciliter. The age at which control was lost was the best, and frequently the only, predictor of the childs IQ at the age of 8 or 10 years and of the deficit in the childs IQ as compared with those of his or her unaffected siblings or parents. The age at which control was lost was also the best predictor of the deficit in scores on the Wide Range Achievement Test of children with PKU at the age of eight, as compared with their unaffected siblings, and of behavior problems (Louisville Behavior Checklist). Variables that were not significant included the maximal phenylalanine concentration before dietary therapy, the age at which treatment was started, and the familys coping abilities. The greatest deficiencies in all of these outcomes were observed among children who were out of dietary control before the age of six years. The highest correlation between the IQs of the children with PKU and their respective parents was observed among the children still on the diet at the age of eight years; the lowest, among those who had poor dietary control before the age of six. These findings suggest that phenylalanine restriction should continue after the age of eight years in children with PKU.

Improving response rates through incentive and follow-up: The effect on a survey of physicians' knowledge of genetics

OBJECTIVES This study assessed efforts to increase response rates to a mailed physician survey and examined whether, as a result, nonresponse bias was reduced. METHODS Randomly selected physicians and geneticists were mailed a questionnaire concerning genetics knowledge and attitudes. In the final but not the pilot survey, a

A randomized trial of oral iron on tests of short-term memory and attention span in young pregnant women

25 incentive and intensive follow-up were used to increase the response rate. RESULTS The response rate from physicians in the final survey was 64.8% (n = 1140), compared with 19.6% in the pilot test (n = 69). Sample representatives in sociodemographic and practice characteristics was improved by follow-up. Respondents recruited with more difficulty did not differ on the principal outcome variable, genetics knowledge, except on one subscore. Pilot study and final survey respondents did not differ in knowledge. CONCLUSIONS Although the effect of increased response rates on the principal outcome variable in this study was minimal, this may not be the case for other studies. Every effort should be made to attain as high a response rate as is practical and to establish that respondents are representative of the population being sampled.

Genetic testing and public policy

Recent studies suggest that infant behavior and psychological test performance are impaired by iron deficiency and may be improved by iron. Comparable studies have not been performed in older populations. Young women early in pregnancy whose nutritional intake may be impaired by poverty constitute a high-risk population. Women aged 14-24 years coming for prenatal care at or before 16 weeks gestation whose hematocrits were greater than or equal to 31% were randomized in a double-blind trial to receive vitamins supplemented with iron (experimental group) or vitamins alone (controls). Hematologic status and tests of short-term memory and attention span were assessed at entry and conclusion of the one-month treatment period. The experimental group showed significant improvement on the most sensitive measure of short-term memory and three subtests. On comparison of the change between initial and final scores, the experimental group showed significant or borderline greater improvement than controls on three tests. These results indicated a beneficial effect of iron therapy on psychometric test-score performance.

Prenatal Genetic Testing: Content of Discussions Between Obstetric Providers and Pregnant Women

This is the last of four articles discussing the broader implications of advances in genetics The increasing rate of discovery of genes related to disease and the development of tests for them has fostered the idea that healthy people will be able to prevent future disease by undergoing genetic testing. The genetics of many diseases are such that tests have only a limited ability to predict the clinical outcome accurately. Nevertheless, the benefits of some predictive genetic tests can be substantial, such as screening newborns for phenylketonuria and sickle cell anemia and testing of older children at risk of familial medullary thyroid carcinoma and multiple endocrine neoplasia type 2a.1 The fact remains that relatively few interventions have yet been devised to improve the outcome of most mendelian disorders.2 When no treatments are available, genetic testing can be used to avoid the conception or birth of affected offspring. Carrier screening in Sardinia, Cyprus, and elsewhere in the Mediterranean has led to an appreciable reduction in the birth of infants with thalassaemia through the use of prenatal diagnosis in couples at high risk and their selective termination of affected fetuses.3 Genetic testing (box), including prenatal testing, also carries the risks of inducing psychological sequelae4 and of making individuals vulnerable to discrimination and diminishing their privacy. Additional benefits of testing in refining clinical diagnosis and tailoring treatent may be in the offing,5 but these remain to be shown. Both the American and British governments have recognised at least some of these problems, but solutions have been slow in coming. The United States started earlier to create a structure to consider the problems, but at present no federal committee is dedicated to addressing issues of genetic testing (box). In its final report, the Task Force on Genetic Testing reviewed …

Mapping the human genome: an assessment of media coverage and public reaction.

Objective To document the content and accuracy of discussions about prenatal genetic testing between obstetric providers and pregnant women. Methods The first prenatal visits of 169 pregnant women with 21 obstetricians and 19 certified nurse-midwives were audiotaped and analyzed for whether a discussion of family history or genetic testing took place and if so, its length, content, and accuracy. Results Family history was discussed in 60% of visits, maternal serum marker screening in 60%, second-trimester ultrasonography for fetal anomalies in 34%, and for women at least 35 years old, amniocentesis or chorionic villus sampling (CVS) in 98%. The length of discussions of genetic testing averaged 2.5 minutes for women younger than 35 years of age and 6.9 minutes for older women. Topics discussed most often were the practical details of testing, the purpose of testing, and the fact that testing is voluntary. Discussions seldom were comprehensive. Obstetricians were more likely to make a recommendation about testing than were nurse-midwives and were less likely to indicate that testing is voluntary. Most women were satisfied with the amount of information, and the majority of women of advanced maternal age had made a decision about amniocentesis or CVS by the end of the visit. Conclusion The information about genetic testing provided in the first prenatal visit is inadequate for ensuring informed autonomous decision-making. Guidelines addressing the content of these discussions should be developed with input from obstetricians, nurse-midwives, genetic counselors, and pregnant women.

Hyperphenylalaninemia due to dihydropteridine reductase deficiency. Assay of the enzyme in fibroblasts from affected infants, heterozygotes, and in normal amniotic fluid cells

Purpose: To assess public reactions to the June 26, 2000, announcement that scientists had nearly finished mapping the human genome.Methods: We conducted a random-digit telephone survey of 407 Maryland residents as well as a content analysis of 55 relevant media reports.Results: African Americans were more likely than Caucasians to report a negative reaction (P < 0.001) to the genome announcement. Overall, privacy/discrimination (16%) and human cloning (14%) were the most commonly mentioned concerns regarding the impact of the genome mapping.Conclusions: These findings highlight the need for continued public discourse, including through the media, to address concerns regarding the Human Genome Project.

ACMG recommendations on incidental findings are flawed scientifically and ethically.

Two infant siblings with modest elevations of serum phenylalanine concentrations had seizures and developmental regression: they died in their second year. Dihydropteridine reductase activity, which can be measured in normal cultured skin fibroblasts, was measured in the younger sibling and was absent. Parents of the two siblings and parents of a previously reported patient all showed 50% or less of the normal dihydropteridine reductase activity in their cultured fibroblasts. Dihydropteridine reductase activity is also present in normal cultured amniotic fluid cells, offering the possibility of prenatal diagnosis. Absence of dihydropteridine reductase results not only in a defect in the conversion of phenylalaning to tyrosine, but also in the biosynthesis of the neurotransmitters, dopamine, norephinephrine, and serotonin. Since deficiencies in these neurotransmitters would not be alleviated by a phenylalanine-restricted diet, it is important to establish the nature of the enzymatic defect in all suspected variants of phenylketonuria.

“Decoding” Informed Consent: Insights from Women regarding Breast Cancer Susceptibility Testing

To the Editor: The “ACMG Recommendations for Reporting of Incidental Findings in Clinical Exome and Genome Sequencing”1 maintain that patients who consent to have their own or their child’s whole genome or exome sequenced should be informed of certain incidental findings (n = 57) “without reference to patient preferences.” A recent supporting paper noted, “In the event of malpractice litigation, the recommendations may be introduced as evidence of the standard of care.”2 This is premature because the recommendations are flawed scientifically as well as ethically. They are based on the beliefs that the presence of these incidental findings indicates that the patient or his/her relatives will suffer future harm and that interventions are available to reduce or prevent harm. The evidence to support these beliefs is insufficient to constitute reporting them as “the standard of care.” The 57 incidental findings listed in the American College of Medical Genetics and Genomics (ACMG) recommendations were derived from the opinions of 16 clinical geneticists and/or molecular laboratory directors3 and “supplemented by a provisional list of genes.”4 The 16 raters “were told to assume that the sequencing was perfectly accurate ... even though this degree of accuracy is not available through current WES/WGS [whole-exome sequencing/whole-genome sequencing] technologies.”3 Each finding is a sequence variation that has been “previously reported and is a recognized cause of the disorder” or is a previously unreported sequence “of the type expected to cause the disorder.” Of the 57 findings, 46 were rated in the study by Green et al.3 Two more (Ehlers–Danlos; catecholaminergic polymorphic ventricular tachycardia) were rated as “Bin 1” (reportable) by Berg et al.4 (I could not find the source for the nine remaining findings for which reporting is mandatory. They are WT1related Wilms’ tumor (one gene), neurofibromatosis type 2 (one gene), arrhythmogenic right-ventricular cardiomyopathy (five genes), and malignant hyperthermia susceptibility (two genes).) In only 14 of the 46 incidental findings rated in the study by Green et al.3 did all raters agree that the finding in both adults and children should be reported; in 12 others, 100% consensus was attained for reporting their presence in adults only. Agreement to report was lower when raters were asked to rate previously unknown truncating and missense variants. When experts disagree, how can the reporting of these findings be mandatory? The previously reported sequence variations have been found in families in which at least one individual expresses the disease phenotype. But as incidental findings, the sequence variations are likely to be found in individuals without a family history. We do not know if any of the reportable variations will have as high penetrance in such families as in those in whom the disease phenotype is present; there could be modifying genes or gene copies that reduce penetrance. The same is even more likely for previously unreported variations for which no genotype–phenotype relation has been established. Somewhat cryptically, Green et al.1 acknowledge these problems: “As additional evidence accrues on the penetrance of these variants among persons without symptoms or family history, these recommendations will be expected to evolve.”1 I interpret this to mean that the incidental presence of some of these variations could be false positives; individuals will then have been compelled to receive erroneous information. With such uncertainty, it seems wise to (i) treat the reporting of “pathogenic” sequence variations as research; (ii) give patients who consent to whole-genome sequencing/ whole-exome sequencing the opportunity to consent to being informed, or not, of incidental findings, having been told of the possible benefits and the unknown probability of false positives (and false negatives); and (iii) have patients who consent to being informed of incidental findings enrolled in a data registry for the purpose of collecting evidence of a genotype–phenotype relationship and whether the expected phenotype can be prevented or ameliorated by intervention. The ACMG recommendations do not include reporting to a data registry, although the authors acknowledged “a great need to develop a central repository of genotypic and phenotypic data.”5 What is the rush to mandate informing patients, or their providers, of incidental findings before “the benefits, harms and costs that may result”1 have been established? Green et al.1 have either not heard of the precautionary principle or have chosen to disregard it. That is, perhaps, the most unethical aspect of the ACMG recommendations. There are additional uncertainties. Lam et al.6 found that the two most frequently used sequencing platforms are not in perfect agreement in detecting variations. For example, “We detected 444,678 SNVs (single-nucleotide variants) by only one platform or the other but not both.” Although confirmatory techniques, such as Sanger sequencing, can reduce or eliminate analytical false positives, they cannot eliminate analytical false negatives. The ACMG should put the horse before the cart and work toward improving the reliability of sequencing in clinical laboratories, as well as toward laboratory quality control and ensurance of appropriate genetic counseling, before making pronouncements on incidental findings in clinical practice. Green et al.1 invert the importance of returning incidental findings in research as compared with practice. “There is,” they say, “an active debate about the return of incidental findings