PKU and NON-PKU Hyperphenylalaninemia: Differentiation, Indication for Therapy and Therapeutic Results

The differential diagnosis of phenylalanine hydroxylase deficiency (PAHD) by biochemical methods is difficult. Using standardized oral protein loading or the intravenous deuterated phenylalanine (phe) load in 46 patients with PAHD, three groups could be distinguished: 1) Phenylketonuria (PKU) with plasma phe levels over 20 mg% under the protein load and with residual activities of less than 1% of normal; 2) mild PKU with plasma phe of 10–20 mg% and residual enzyme activities of 1–3%; 3) Non-PKU hyperphenylalaninemia with plasma phe levels of less than 10 mg% and residual enzyme activities of more than 3%. Psychomotor development in 32 untreated patients with PAHD showed that there is a high risk of brain damage for all patients with in vivo residual activities of less than 3% of normal. Restriction fragment length polymorphism (RFLP) haplotypes at the phenylalanine hydroxylase (PAH) locus in 60 German patients with PAHD showed that 90% of the mutant alleles are confined to four distinct haplotypes. Using an oligonucleotide probe for the splicing mutation associated with mutant haplotype 3 a close association between the mutation and the haplotype 3 has been observed. There is also an association between haplotypes 2 and 3 and PKU patients with residual enzyme activities of less than 1%. However, in only 37% of our patients with PAHD could a direct diagnosis of the mutations in the PAH gene be made. More knowledge of other mutations in the PAH gene is necessary to differentiate patients with PAHD on the DNA level.     

Direct diagnosis of predominant mutation delta F508 associated with the mucoviscidosis gene

The cystic fibrosis locus was mapped on the long arm of the chromosome 7 in 1985. It has recently been cloned and a three base pair deletion has been recognized as the mutation associated with the majority of CF chromosomes (delta F508). CF haplotypes previously defined with tightly associated DNA markers were analysed using PCR (Polymerase Chain Reaction) and allele specific oligonucleotides to determine the presence or absence of this mutation. This mutation was found on 80% of our CF chromosomes and associated predominantly with the B haplotypes. The detection of this mutation is now a major improvement for carrier detection and prenatal diagnosis of the disease.     

Evaluation of the usefulness for neonatal mass screening in light of 35 years personal experience

The results and the significance of neonatal mass-screening programmes for inborn errors of metabolism, conducted by the National Research Institute of Mother and Child (NRIMC), are discussed. As the first in Poland, in 1964, mass-screening for phenylketonuria (PKU) was introduced. The BIA-Guthrie test was used. Other Guthrie tests (GBIA) were applied in homocystinuria, tyrosinemia, histidinemia and leucinosis (Maple Syrup Urine Disease-MSUD). In the middle of the 60. the Beutler and Baluda test was introduced for galactosaemia, as well as the Efron urine test in infant screening for different inborn errors of metabolism. In the middle of the 70., neonatal mass-screening for cystic fibrosis (CF, mucoviscidosis) was started. Meconium tests and the sweat test with ion selective chloride electrode were used. Apart from inborn errors of metabolism, we also introduced a screening programme for neuroblastoma in which vaniline mandelic acid (VMA) in urine was estimated and for congenital hypothyroidism were TSH level was assessed. The results of screening are shown in the tables and in the figures. In our opinion the best clinical results are obtained with screening for congenital hypothyroidism and for PKU, since very early detection and treatment in these diseases prevents severe mental retardation. We therefore consider that both these screening programmes should be treated as obligatory examinations in all neonates. Taking into consideration the fact that there are different types of hyperhenylalaninemias, the principles of differential diagnosis are discussed. Molecular genetic investigations, carried out in the NRIMC Department of Genetics proved to be a very important procedure in the verification of diagnosis of different mutations. The authors also discuss the problem of dietary treatment duration in PKU. In our opinion the hypophenyloalanine diet regimen in girls, should not be discontinued during adolescence, since there is the problem of maternal PKU and the possibility of foetal damage. The results of our own investigations of maternal PKU are discussed. The significance of mass-screening for galactosemia is still under discussion. In our opinion, mass-screening for galactosemia is not useful and we have discontinued it. Selective screening has been started combined with molecular genetic studies in high risk families. In the future, we plan to prepare guidelines on the principles of diagnosis and treatment of galactosemia in children and women in the reproductive age. Mass-screening for cystic fibrosis is also still under discussion. The results of the early screening programmes were not satisfactory and the tests were discontinued. In 1998, after reorganisation of the whole system, CF screening, using tripsin-radioimmune assays, was started again. The new screening programme is combined with molecular genetic investigation of different mutations. It is still too early to assess the importance and success of this CF mass-screening programme. We decided to discontinue the screening for homocystinuria, histidinemia, tyrosinemia, leucinosis and for neuroblastoma, since these programmes did not comply with criteria of mass-screening. In 1997, major reorganisation of screening programmes for inborn errors of metabolism, at NRIMC, was undertaken. The Guthrie test for PKU was changed to a quantitative colorimetric method. The immuno-luminometric method is used for TSH estimation. The whole system is based on complete computer control of all the steps of screening, from blood sampling on filter paper until the final diagnosis. The advantages of this modern system of organisation of the screening programme are discussed.     

Genetic testing for cystic fibrosis.

Cystic fibrosis occurs in 1 in 2000 to 4000 whites, and about 1 in 25 people are heterozygous carriers. The CF gene is cloned, and a single common mutation is found on 70% to 75% of CF chromosomes in most populations. Numerous different mutations are found on the remaining CF chromosomes. DNA analysis for carrier testing should be offered to relatives of CF patients and to reproductive partners of proven carriers. Population based carrier screening could detect about 72% of at-risk couples, and pilot studies are in progress to assess the feasibility of such screening. With regard to these factors, the possibility of gene therapy, and the questions raised, CF serves as a useful model for many genetic disorders.     

Surfactant protein A and B genetic variants and respiratory distress syndrome: allele interactions

The contribution of multiple genetic components in disease pathogenesis is relevant to both diseases of multifactorial and/or multigenic etiology such as the respiratory distress syndrome (RDS) and to diseases where a single gene has been identified as the disease-causing gene. An example of the latter is cystic fibrosis (CF) where the disease-causing gene has been clearly identified as the CF transmembrane conductance regulator gene, but genetic variants of the mannose binding protein and surfactant protein A have been associated with disease severity in CF. The overall rationale for considering genetic contribution to disease pathogenesis is based on the premise that all diseases or deaths (except perhaps those resulting from trauma) have a genetic component. The difference in genetic contribution among various diseases is the percent contribution and the number of factors that make this contribution. Therefore, if the number of genetic contributors is small and the percentage of genetic contribution is high it may be less challenging to identify such factors. In this paper we summarize allele associations and discuss allele interaction of the surfactant protein genes in relation to RDS (the term allele and genetic variant will be used interchangeably).     

Mutation analysis in the diagnosis of cystic fibrosis

Since the characterization of the gene encoding the cystic fibrosis transmembrane conductance regulator protein and identification of its main mutation, F508, causing cystic fibrosis (CF), more than 150 mutations in this gene have been reported, most of them only in a few or even single CF patients. Attempts to use mutation analysis in genetic counselling or for the diagnosis of CF depends on prevalence data of certain mutations in the respective population, and considerable ethnic differences have been reported. In this study we determined the prevalence of the mutations F508, G551D, R553X, and G542X and of genotypes defined by these mutations in 239 CF patients (444 independent CF chromosomes) seen in our clinic. The analysis for those four mutations alone now permits identification of approximately 75% of all mutations in our CF patients. The complete genotype can be resolved in approximately 63% of patients. This represents the diagnostic sensitivity which can be achieved by mutation analysis in patients without a family history of CF. We conclude that in situations where conventional diagnostic tests are not feasible or difficult to interpret, mutation analysis using a limited set of mutations can contribute significantly to an early and specific diagnosis of CF.     

Spectrum and origin of phenylketonuria mutations in Spain

In order to characterize the molecular heterogeneity of phenylalanine hydroxylase deficiencies in the Spanish population, 37 PKU patients were initially screened for 16 known European mutations. For the remaining unidentified alleles, we used a combined strategy based on single strand conformation polymorphism analysis and DNA sequencing. Overall, a total of 15 different mutations were found in our sample, which account for 62% of the total mutant alleles. We also investigated the association between the mutations, haplotypes and variable number of tandem repeats described on the phenylalanine hydroxylase gene. In addition, we analyzed the geographical distribution in Spain of the two most prevalent mutations in our population: IVS10 and I65T.     

Novel mutation in the epithelial sodium channel causing type I pseudohypoaldosteronism in a patient misdiagnosed with cystic fibrosis

Cystic fibrosis (CF) is an inherited disorder with a devastating prognosis. Determination of chloride concentration in sweat has been the gold standard test for diagnosing CF for over 50?years and still remains the primary screening test. However, now that the genetic cause is known and can be studied, genetic confirmation is mandatory in every suspected patient. We present a patient who had been clinically diagnosed and whose genetic testing could not confirm CF, leading us to search for other options that may also give a positive sweat test. The patient turned out to suffer type 1 pseudohypoaldosteronism, a condition that may cause severe dehydration, hyponatremia and hyperkalemia episodes if not diagnosed and treated early with sodium supplementation. We found a genetic variation in the epithelial sodium channel gene which has not been reported previously, and we discuss the possibility of it being the cause of our patient's phenotype. Conclusion: this patient clearly illustrates the usefulness of genetic confirmation for CF for the diagnosis and genetic counselling, even when it is clinically oriented, and describes a novel mutation of the amiloride-sensitive epithelial sodium channel possibly causing type 1 pseudohypoaldosteronism.     

宁夏苯丙酮尿症患儿苯丙氨酸羟化酶基因外显子7突变分析

目的 探讨宁夏地区苯丙酮尿症（PKU）患儿苯丙氨酸羟化酶（PAH）基因外显子7 突变类型及频率,为该地区PKU 的基因诊断和产前诊断提供依据。方法 应用PCR 产物直接测序方法,对宁夏73 例经典型PKU 患儿（回族39 例,汉族34 例）的146 个PAH 等位基因外显子7 及其旁侧内含子区域进行序列分析。结果 共检测出6 种突变基因型,分别是R243Q（14.4%）、R241C（6.8%）、IVS7+2T → A（2.7%）、L255S（0.7%）、G247V（0.7%）和G247R（0.7%）。外显子7 突变基因总频率为26.0%（38/146）,包括错义突变和剪接位点突变两种。回族患儿R241C 等位基因突变检出率高于汉族（10% vs 3%,P<0.05）。结论 宁夏地区PKU 患儿PAH 基因外显子7 突变频率最高的是R243Q,其次为R241C;回族和汉族PKU 患儿R241C 等位基因突变率不同。     

The cystic fibrosis defect approached from different angles — new perspectives on the gene,the chloride channel,diagnosis and therapy

The search for the basic defect in cystic fibrosis (CF) has reached a decisive stage since the recent identification of the responsible gene. Electrophysiological and biochemical research had defined the CF defect as a dysregulation of epithelial chloride channels. The putative protein product of the now identified gene shares properties with other known transport proteins, but it is not necessarily itself a chloride channel protein. Elucidation of the primary cellular defect will certainly have important aetiological and hopefully therapeutic implications. The identification of the major gene mutation already has significant consequences for genetic counselling and prenatal diagnosis. Heterozygote detection at the population level awaits identification of the probably heterogenous mutations on about 30% of the CF chromosomes. At present, about 50% of CF patients are homozygous for the recently identified major CF mutation.     

Experience and Problems of Newborn Mass Screening for Inborn Errors of Metabolism in Japan

A program of newborn mass screening for inborn errors of metabolism has since 1977 been conducted by The Ministry of Health and Welfare in Japan with great success in preventing mental and physical handicaps in children and estimating the incidence of these diseases in our country as follows: phenylketonuria (PKU) 1/78,100 maple syrup urine disease (MSUD) 1/281,200 histidinemia 1/10,600 galactosemia 1/156,200 There are some genetic variants of each of these diseases, PKU, MSUD and others. Circumspection is therefore required in making a diagnosis or selecting a particular method of treatment. When MSUD is suspected, it is necessary to make a definite diagnosis promptly and take therapeutic measures including peritoneal dialysis. The efficacy of low histidine diet in histidinemia is not yet ascertained. Close inquiries should be made about the benefit of such a dietary regimen and the long term prognosis therewith. A screening test for homocystinuria by the detection of blood methionine level is difficult. In cases with high blood methionine level during the neonatal period, it is necessary to differentiate the condition from other disease with high blood methionine. Hepatorenal tyrosinemia appears to be unsuitable for mass screening because of the the unefficacy of low phenylalanine tyrosine diet in some of the patient and the difficulty to differentiate from transient tyrosinemia in normal newborn infants. However, mass-screening is recommendable for Richner-Hanhalt type tyrosinemia which responds well to dietary therapy and which is easy to differentiate from the transient tyrosinemia because of the marked elevation of blood tyrosine.     

Direct detection of a major mutation responsible for phenylketonuria in the population of the Federal Republic of Germany

Forty-six individuals having phenylketonuria (PKU) alleles at the phenylalanine hydroxylase (PAH) locus were tested for the haplotype 2 PKU mutation by allele-specific hybridization following in vitro DNA amplification. Patients and carriers previously shown to have a mutant haplotype 2 PAH allele demonstrated conservation of this mutation. In vitro DNA amplification greatly facilitated this analysis and provides the possibility of population screening for 37% of the mutant German PAH alleles.     

Prenatal screening for phenylketonuria in 2 families by trophoblast biopsy

Prenatal diagnosis of phenylketonuria (PKU) is now available owing to restriction fragment length polymorphism (RFLP) of the phenylalanine hydroxylase gene. Biopsies of chorionic villi were carried ou at 11 weeks gestation in 2 families who had previously a child with the classical form of PKU and who were considered informative after DNA studies. Fetal DNA was extracted and studied with restriction enzymes selected according to the study of each family. Hybridization studies suggested that one fetus was affected by the disease and that the second was normal. Termination of pregnancy was carried out in the first case; however, study of the fetus could not be performed. In the second family, the pregnancy resulted in the delivery of a normal child, as shown by normal phenylalanine level at birth.     

Family studies of the steroid 21-hydroxylase and coplement C4 genes define 11 haplotypes in classical congenital adrenal hyperplasia in The Netherlands

Two steroid 21-hydroxylase genes are normally present within the human major histocompatibility complex near the genes encoding the fourth component of complement (C4A and C4B). Steroid 21-hydroxylase is encoded by the CYP21 gene, while the highly homologous CYP21P gene is a pseudogene. We studied steroid 21-hydroxylase and complement C4 haplotypes in 33 Dutch patients (29 families) suffering form classical congenital adrenal hyperplasia (CAH) and in their 80 family members, and also in 55 unrelated healthy controls, using 21-hydroxylase and complement C4 cDNA probes. Eleven different haplotypes, defined in terms of gene deletions, gene duplications, conversions of CYP21 to CYP21P, and long and short C4 genes, were found. In 23% of the patients' haplotypes, the CYP21 gene was deleted; in 12%, it was converted into a CYP21P pseudogene. In the remaining 65%, the defect was apparently caused by a mutation not detectable by this method. The most common haplotype (with one CYP21 and one CYP21P gene) was significantly more often observed in patients with simple virilizing CAH than in those with salt-losing CAH. Comparison of the 21-hydroxylase haplotypes found in CAH patients from several countries shows evidence for considerable genetic variation between the groups studied.     

云南苯丙氨酸羟化酶基因内(TCTA)n多态性及其在苯丙酮尿症诊断中的应用

目的应用苯丙氨酸羟化酶（ＰＡＨ）基因内（ＴＣＴＡ）ｎ多态性连锁分析进行经典型苯丙酮尿症（ＰＫＵ）的基因诊断和产前诊断。方法应用聚合酶链反应扩增片段长度多态性（ＰＣＲＡｍｐＦＬＰ）方法,分析云南省１３个家系苯丙氨酸羟化酶（ＰＡＨ）基因内（ＴＣＴＡ）ｎ多态性。结果在１３个家系中检测到２２４～２５２ｂｐ的８种等位片段,其ＰＩＣ为０．６９８,杂合频率是５１％。可诊断率为１００％和５０％的家系各６个,１个家系因双亲带型为纯合型而未能诊断,可诊断率为６９％。完成１例产前基因诊断和２例回顾性的基因诊断。结论（ＴＣＴＡ）ｎ的ＰＣＲＡｍｐＦＬＰ分析可作为经典型ＰＫＵ基因诊断和产前诊断的一种简便有效的方法。     

Molecular detection of genetic defects in congenital adrenal hyperplasia due to 21-hydroxylase deficiency: A study of 27 families

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase (21-OHase) deficiency is inherited as an autosomal recessive trait. Patients can present with the salt wasting, simple virilizing or a non-classical form of the disease. The gene for P450C21, the enzyme carrying 21-OHase activity, has been mapped to the major histocompatibility complex on chromosome 6p. Using molecular hybridisation techniques we have studied the genetic defect in 27 families with one or more affected off-spring diagnosed and treated at the University Hospital of Essen. DNA samples were digested with restriction endonucleaseTaqI,PvuII,BglII, andEcoRI and analysed by Southern blot hybridisation with the cDNA probe pC21/3c. Eleven of 40 haplotypes associated with the salt wasting form were found to have a large deletion of 30 kb affecting the 5 end of the active 21-OHase gene and the 3 end of the closely linked pseudogene. Results in another 11 cases are compatible with gene conversion; 18 cases were not informative. The 30 kb deletion was associated with a combination of the HLA antigens Bw47 and DR7 in 7 of 11 cases. In the haplotypes with gene conversion, no linkage disequilibrium to HLA antigens was found. No apparent gene alterations were detected in simple virilizing and non-classical haplotypes. The direct detection of the genetic defect in 55% of the salt wasting haplotypes may help to improve predictive testing in families with CAH.     

Population genetics of phenylketonuria

Phenylketonuria (PKU) is an autosomal recessive disorder caused by a large number of mutations at the phenylalanine hydroxylase (PAH) locus, most of which are strongly associated with specific RFLP or VNTR haplotypes. One of the major questions remaining in PKU research is why this apparently maladaptive disorder has been maintained at a frequency of approximately 1 in 10000 among Caucasians. A growing number of studies have provided evidence that both the relatively high frequency of PKU and the strong mutation/haplotype associations might reflect the existence of multiple founding populations for PKU. Examples of putative founding populations for PKU in both Europe and Asia will be presented. Some PAH mutations are associated with multiple haplotypes, suggesting recurrence. Evidence for and against recurrence as the mechanism responsible for the association of the R408W mutation with RFLP haplotypes 1 and 2 will be discussed.     

Mutation screening for prenatal and presymptomatic diagnosis: cystic fibrosis and haemochromatosis

Hereditary haemochromatosis (HH) and cystic fibrosis (CF) are the most common autosomal recessively inherited disorders in Caucasian populations. In its typical form, CF manifests during the first years of life, while the mean age of onset of organ damage is 54 years in HH. Both disorders can be diagnosed presymptomatically utilising biochemical and/or genetic testing. Since approximately 90% of mid-European HH patients are homozygous for only one specific mutation (C282Y) in the candidate gene for HH, genetic testing is simple and sensitive in HH. In CF, molecular testing is currently hampered by the large number (more than 800) of mutations in the CF transmembrane conductance regulator gene. Several studies have been initiated to investigate the potential benefits and the best time and mode of presymptomatic testing for HH and CF. Conclusion Mutation analysis is widely recommended for presymptomatic diagnosis of cystic fibrosis and hereditary haemochromatosis because of the presumed benefit, although several medical, ethical, social and technical questions warrant further investigations. Prenatal mutation testing is commonly performed for cystic fibrosis but not for hereditary haemochromatosis. Informed consent of tested individuals and the availability of genetic counselling is a prerequisite for any mutation screening approach in either disease.     

Evaluation of 47,213 infants in neonatal screening for cystic fibrosis, using pancreatitis-associated protein and immunoreactive trypsinogen assays]

OBJECTIVES: The increasing evidence of the benefits of neonatal screening for cystic fibrosis (CF) indicates that this procedure could soon be implemented throughout France. The screening strategy currently used involves the detection of infants with elevated levels of immunoreactive trypsinogen (IRT) (approximately 1% of the population), followed by the detection of CFTR gene mutations. However, genetic analysis has certain drawbacks, the most important of which being the management of heterozygotes, and in France the requirement by law of previous informed consent. In cases of CF, pancreatic alterations are already present in utero. A previous study has demonstrated the value of pancreatitis-associated protein (PAP) as a screening test for CF, and has indicated that a feasible two-stage strategy could involve the following: 1) selection of infants with elevated PAP levels; 2) in this group of infants, subsequent detection of those with elevated IRT levels for direct CF diagnosis by the sweat test thereby avoiding the use of genetic analysis. The study aim was to evaluate this strategy in a large number of neonates. METHODS AND RESULTS: The aforementioned strategy was evaluated in a prospective study involving 47,213 infants in the Provence region of France. In infants with a PAP > 7.5 ng/mL (1.28%), 176 had an elevated IRT level > 700 ng/mL (0.37%). In this limited population sample (0.37% of the total), the sweat test diagnosed five cases of CF. A sixth case involving the monozygous twin of an infant with diagnosed CF remained undetected, probably because of a registration error. Genetic analysis confirmed the diagnosis, and also detected another case in an infant with two CFTR mutations but with a normal phenotype at 20 months of age. As the observed incidence was similar to that which had previously been reported, and as no further case was subsequently detected two years after the end of the study, this indicated that the sensitivity of this screening strategy was satisfactory. Its specificity makes the direct diagnosis of CF cases by the sweat test feasible, without further selection by genetic analysis. CONCLUSION: The PAP/IRT technique for CF detection seems to be suitable for mass screening, without the drawbacks of genetic testing.