Compound heterozygosity of two functional null mutations in the ALPL gene associated with deleterious neurological outcome in an infant with hypophosphatasia.

Hypophosphatasia (HPP) is a heterogeneous rare, inherited disorder of bone and mineral metabolism caused by different mutations in the ALPL gene encoding the isoenzyme, tissue-nonspecific alkaline phosphatase (TNAP). Prognosis is very poor in severe perinatal forms with most patients dying from pulmonary complications of their skeletal disease. TNAP deficiency, however, may also result in neurological symptoms such as neonatal seizures. The exact biological role of TNAP in the human brain is still not known and the pathophysiology of neurological symptoms due to TNAP deficiency in HPP is not understood in detail. In this report, we describe the clinical features and functional studies of a patient with severe perinatal HPP which presented with rapidly progressive encephalopathy caused by new compound heterozygous mutations in the ALPL gene which result in a functional ALPL "knock out", demonstrated in vitro. In contrast, an in vitro simulation of the genetic status of his currently asymptomatic parents who are both heterozygous for one mutation, showed a residual in vitro AP activity of above 50%. Interestingly, in our patient, the fatal outcome was due to progressive encephalopathy which was refractory to antiepileptic therapy including pyridoxine, rather than hypomineralization and respiratory insufficiency often seen in HPP patients. The patient's cranial MRI showed progressive cystic degradation of the cortex and peripheral white matter with nearly complete destruction of the cerebrum. To our knowledge, this is the first MRI-based report of a deleterious neurological clinical outcome due to a progressive encephalopathy in an infant harboring a functional human ALPL "knock out". This clinical course of disease suggests that TNAP is involved in development and may be responsible for multiple functions of the human brain. According to our data, a certain amount of residual TNAP activity might be mandatory for normal CNS function in newborns and early childhood.

Functional characterization of a novel mutation localized in the start codon of the tissue-nonspecific alkaline phosphatase gene.

Hypophosphatasia (HPP) is a rare inborn disease caused by different mutations in the tissue-nonspecific alkaline phosphatase (ALPL) gene. Previous studies showed that gene mutations could exhibit a dominant negative effect leading to a mild HPP phenotype in heterozygous carriers. In the present report we describe the clinical and functional studies of a novel mutation localized in the start codon of transcript variant 1 of the ALPL gene from a female adult heterozygous carrier. The mutation results in translation of an N-terminally truncated protein, which might be identical to the deduced protein from ALPL transcript variant 2. When overexpressed in HEK-293 cells it does not exhibit any enzymatic activity and has no significant effect on the wild type ALPL protein. Furthermore it is not attached to the cell membrane. Due to the loss of the signal peptide an intracellular misrouting and a premature degradation is obvious. Hence the new isoform deposited in the database does not produce an active protein as it is the case in the natural mutation of our patient. Since the mutation does not produce a dominant negative protein in heterozygous carriers, the clinical phenotype in our patient and her relatives is very mild with only unspecific myalgia. However the patient developed bone marrow edema of both femoral heads during lactation after delivery of a healthy child, indicating a risk to develop alterations of bone metabolism in challenge situations. Her sister complains of identical symptoms, her father shows distinct symptoms of odonto-hypophosphatasia. The question if or if not carriers of ALPL mutations in general or only with distinct genotypes can be symptomatic in normal life or in challenge situations requires systematic clinical studies.

The exon-intron organization of the prohormone convertase PC2 gene from the insect Lucilia cuprina.

Prohormone or proprotein convertases are members of the subtilisin family of serine proteases. They are involved in the activation of precursor molecules by endoproteolytic cleavage at basic amino acid residues. Among the different members of this prohormone convertase family, the prohormone convertase 2 (PC2) is almost exclusively expressed in endocrine and neuroendocrine tissues and plays an important role in the endoproteolytic processing of prohormones. Here we describe the exon-intron organization of the PC2 gene from the insect Lucilia cuprina by characterization of PCR-amplified genomic DNA fragments. The insect PC2 gene contains 12 exons with an estimated size of over 14.5 kb. The exon sizes range from 38 bp to > 448 bp. All identified intron-exon boundaries are consistent with the GT-AG-rule. A comparison of the genomic structures of the thus far known prohormone convertase genes with that of the insect PC2 gene revealed a conservation of the positions of most introns interrupting the exons coding for the amino-terminal and catalytic domains. This conservation is consistent with the suggestion of a common evolutionary origin for the prohormone convertase gene family.

Isolation and characterization of insect PC2-like prohormone convertase cDNA.

Endoproteolytic processing of large precursor molecules at basic amino acid residues plays an important role in the maturation of many hormones, neuropeptides and other regulatory proteins. Enzymes performing these reactions are designated as prohormone or proprotein convertases and belong to the subtilisin family of serine proteases. The screening of a larval cDNA library of the sheep blowfly Lucilia cuprina resulted in the isolation of two cDNAs encoding a PC2-like prohormone convertase. The predicted 675 amino acid preproprotein (LcuPC2) exhibits its highest identity to invertebrate and vertebrate prohormone convertase 2 homologues, and a noticeably lower identity to the so far known insect furin-like prohormone convertases of Drosophila melanogaster and Aedes aegypti. In Northern blot experiments a signal at 2.5 kb could be detected.

Clinical and biochemical investigations and molecular analysis of subjects with mutations in the androgen receptor gene.

OBJECTIVE: Androgen insensitivity syndromes (AIS) in subjects with 46, XY karyotype and normal or even elevated androgen blood levels are characterized by various aberrations in male differentiation and virilization. AIS is often accompanied by a broad spectrum of abnormal binding characteristics of the androgen receptor (AR). In order to investigate the correlation between the degree of virilization defect and the type of androgen binding abnormalities and/or the nature of the mutation in the AR gene, we determined androgen binding characteristics of the AR protein and the sequence of the AR gene in clinically and biochemically well characterized patients with various degrees of androgen resistance. DESIGN AND PATIENTS: The activity of 5 alpha-reductase and the binding of androgen to its receptor (KD-values, Bmax, thermolability) were determined in genital skin fibroblasts from 20 patients with various degrees of defects in virilization (2 CAIS, complete AIS; 18 PAIS, partial AIS patients). The AR gene of these 20 subjects was characterized by PCR-SSCP analysis. In case of aberrant electrophoretic mobility the corresponding exon was sequenced. RESULTS: The 2 patients with CAIS and 7 with PAIS showed a mutation in the AR gene. In two, the mutation was in the DNA binding domain, and in all others in the ligand binding domain. In 11 patients with severe virilization defects no abnormal behaviour was detected in the PCR-SSCP. Transcriptional activation studies of two mutant ARs revealed that an approximately tenfold higher androgen concentration (methyltrienolone) is necessary to achieve maximal response as compared to the wild type AR. CONCLUSIONS: There is no obvious relation between the degree of androgen resistance and the binding parameters of the AR and/or the nature of mutation in the AR gene. Androgen insensitivity syndrome can occur despite normal androgen binding and presumably non-mutated AR genes. Even if there is abnormal binding of androgen and/or a mutation in the AR gene there is no clear-cut relationship between these parameters and the degree of virilization defects. Thus, in a proportion of patients, neither the determination of binding parameters of the AR nor the detection of mutations in the AR gene are sufficient to understand the mechanisms underlying the androgen insensitivity syndrome.