Mutational analysis of the PHEX gene: novel point mutations and detection of large deletions by MLPA in patients with X-linked hypophosphatemic rickets.

X-Linked hypophosphatemic rickets (HYP, XLH) is a disorder of phosphate homeostasis, characterized by renal phosphate wasting and hypophosphatemia, with normal to low 1,25-dihydroxy vitamin D3 serum levels. The purpose of our study was the detection of inactivating mutations in the PHEX gene, the key enzyme in the pathogenesis of XLH. The 16 patients, representing eight families, presented with suspected XLH from biochemical and clinical evidence. All 16 were referred for mutational analysis of the PHEX gene. We detected three novel disease-causing mutations, C59S, Q394X, and W602, for which a loss of function can be predicted. A G28S variation, found in two healthy probands, may be a rare polymorphism. Another mutation, A363 V, is localized on the same allele as the C59S mutation, thus its functional consequences cannot be proven. Furthermore, we detected a deletion of three nucleotides in exon 15 which resulted in the loss of amino acid threonine 535. Heterozygosity of this mutation in a male patient without any chromosomal aberrations suggests its presence as a mosaic. Novel large deletions were detected using multiplex ligation-dependent probe amplification (MLPA) analysis. Two of these deletions, loss of exon 22 alone or exons 21 and 22 together, may result in the translation of a C-terminal truncated protein. Two large deletions comprise exons 1-9 and exons 4-20, respectively, and presumably result in a nonfunctional protein. We conclude that molecular genetic analysis confirms the clinical diagnosis of XLH and should include sequence analysis as well as the search for large deletions, which is facilitated by MLPA.

[The phenylalanine stress test in the classification of patients with phenylketonuria]. / Der Phenylalanin-Belastungstest in der Klassifikation der Phenylketonurie-Patienten.

Classification of defects of phenylalanine hydroxylase is usually performed by a phenylalanine loading test according to Blaskovics. This loading test has some drawbacks, especially negative side effects for the patients. We found a correlation between the result of the loading test and the dietetic phenylalanine tolerance. Therefore the trouble of undergoing the loading rest may be spared many patients.

Newborn screening for hyperphenylalaninemia on day 5: is 240 mumol/liter the most appropriate cut-off level?

In most countries, including the UK, Sweden, Japan, and the FRG, blood samples for newborn screening are not taken until Day 5, in contrast to Day 3 in the United States. This results in fundamentally different conditions with respect to the amount of phenylalanine ingested by the newborn until the day of sampling. Nevertheless those countries who routinely screen on Day 5 have adopted the U.S. cut-off level of 240 mumol/liter (4 mg/dl). This study is the first to compare, via direct evaluation of a regional screening program, whether a higher or a lower cut-off level would be more appropriate for phenylalanine screening on Day 5. In a prospective study on phenylalanine screening with 132,638 infants (cut-off level 120 mumol/liter) the number of recalls was 152. Among them 14 cases with a permanent disorder of phenylalanine metabolism were detected. With the officially recommended cut-off level of 240 mumol/liter as well as with the 360 mumol/liter level used in some centers, the recall rate would have been lower, specificity higher, and the predictive values of a positive result higher. On the other hand sensitivity would have been significantly lower (only 80%). Because in newborn screening the crucial point is sensitivity, our data suggest that in phenylalanine screening done on Day 5 cut-off levels of 240 or 360 mumol/liter are less appropriate than a lower cut-off level. With higher cut-off levels a large proportion of patients (one-fifth) with a permanent disorder of phenylalanine metabolism would be missed.

Rationale for the German recommendations for phenylalanine level control in phenylketonuria 1997.

UNLABELLED: Treatment of hyperphenylalaninaemias due to phenylalanine hydroxylase deficiency with a low phenylalanine (Phe) diet is highly successful in preventing neurological impairment and mental retardation. There is consensus that, for an optimal outcome, treatment should start as early as possible, and that strict blood Phe level control is of primary importance during the first years of life, but for adolescent and adult patients international treatment recommendations show a great variability. A working party of the German Working Group for Metabolic Diseases has evaluated research results on IQ data, speech development, behavioural problems, educational progress, neuropsychological results, electroencephalography, magnetic resonance imaging, and clinical neurology. Based on the actual knowledge, recommendations were formulated with regard to indication of treatment, differential diagnosis, and Phe level control during different age periods. The development of the early-and-strictly-treated patient in middle and late adulthood still remains to be investigated. Therefore, the recommendations should be regarded as provisional and subject to future research. Efficient treatment of phenylketonuria has to go beyond recommendations for blood Phe level control and must include adequate dietary training, medical as well as psychological counselling of the patient and his family, and a protocol for monitoring outcome. CONCLUSIONS: Early-and-strictly-treated patients with phenylketonuria show an almost normal development. During the first 10 years treatment should aim at blood Phenylalanine levels between 40 and 240 micromol/L. After the age of 10, blood phenylalanine level control can be gradually relaxed. For reasons of possible unknown late sequelae, all patients should be followed up life-long.