Genetic analysis of adults heterozygous for ALPL mutations.

Hypophosphatasia (HPP) is a rare inherited metabolic bone disease due to a deficiency of the tissue nonspecific alkaline phosphatase isoenzyme (TNSALP) encoded by the ALPL gene. Patients have consistently low serum alkaline phosphatase (AP), so that this parameter is a good hallmark of the disease. Adult HPP is heterogeneous, and some patients present only mild nonpathognomonic symptoms which are also common in the general population such as joint pain, osteomalacia and osteopenia, chondrocalcinosis, arthropathy and musculoskeletal pain. Adult HPP may be recessively or dominantly inherited; the latter case is assumed to be due to the dominant negative effect (DNE) of missense mutations derived from the functional homodimeric structure of TNSALP. However, there is no biological argument excluding the possibility of other causes of dominant HPP. Rheumatologists and endocrinologists are increasingly solicited for patients with low AP and nonpathognomonic symptoms of HPP. Many of these patients are heterozygous for an ALPL mutation and a challenging question is to determine if these symptoms, which are also common in the general population, are attributable to their heterozygous ALPL mutation or not. In an attempt to address this question, we reviewed a cohort of 61 adult patients heterozygous for an ALPL mutation. Mutations were distinguished according to their statistical likelihood to show a DNE. One-half of the patients carried mutations predicted with no DNE and were slightly less severely affected by the age of onset, serum AP activity and history of fractures. We hypothesized that these mutations result in another mechanism of dominance or are recessive alleles. To identify other genetic factors that could trigger the disease phenotype in heterozygotes for potential recessive mutations, we examined the next-generation sequencing results of 32 of these patients for a panel of 12 genes involved in the differential diagnosis of HPP or candidate modifier genes of HPP. The heterozygous genotype G/C of the COL1A2 coding SNP rs42524 c.1645C > G (p.Pro549Ala) was associated with the severity of the phenotype in patients carrying mutations with a DNE whereas the homozygous genotype G/G was over-represented in patients carrying mutations without a DNE, suggesting a possible role of this variant in the disease phenotype. These preliminary results support COL1A2 as a modifier gene of HPP and suggest that a significant proportion of adult heterozygotes for ALPL mutations may have unspecific symptoms not attributable to their heterozygosity.

Identification of altered brain metabolites associated with TNAP activity in a mouse model of hypophosphatasia using untargeted NMR-based metabolomics analysis.

Tissue non-specific alkaline phosphatase (TNAP) is a key player of bone mineralization and TNAP gene (ALPL) mutations in human are responsible for hypophosphatasia (HPP), a rare heritable disease affecting the mineralization of bones and teeth. Moreover, TNAP is also expressed by brain cells and the severe forms of HPP are associated with neurological disorders, including epilepsy and brain morphological anomalies. However, TNAP's role in the nervous system remains poorly understood. To investigate its neuronal functions, we aimed to identify without any a priori the metabolites regulated by TNAP in the nervous tissue. For this purpose we used 1 H- and 31 P NMR to analyze the brain metabolome of Alpl (Akp2) mice null for TNAP function, a well-described model of infantile HPP. Among 39 metabolites identified in brain extracts of 1-week-old animals, eight displayed significantly different concentration in Akp2-/- compared to Akp2+/+ and Akp2+/- mice: cystathionine, adenosine, GABA, methionine, histidine, 3-methylhistidine, N-acetylaspartate (NAA), and N-acetyl-aspartyl-glutamate, with cystathionine and adenosine levels displaying the strongest alteration. These metabolites identify several biochemical processes that directly or indirectly involve TNAP function, in particular through the regulation of ecto-nucleotide levels and of pyridoxal phosphate-dependent enzymes. Some of these metabolites are involved in neurotransmission (GABA, adenosine), in myelin synthesis (NAA, NAAG), and in the methionine cycle and transsulfuration pathway (cystathionine, methionine). Their disturbances may contribute to the neurodevelopmental and neurological phenotype of HPP.

Molecular diagnosis of hypophosphatasia and differential diagnosis by targeted Next Generation Sequencing.

Hypophosphatasia (HPP) is a rare inherited skeletal dysplasia due to loss of function mutations in the ALPL gene. The disease is subject to an extremely high clinical heterogeneity ranging from a perinatal lethal form to odontohypophosphatasia affecting only teeth. Up to now genetic diagnosis of HPP is performed by sequencing the ALPL gene by Sanger methodology. Osteogenesis imperfecta (OI) and campomelic dysplasia (CD) are the main differential diagnoses of severe HPP, so that in case of negative result for ALPL mutations, OI and CD genes had often to be analyzed, lengthening the time before diagnosis. We report here our 18-month experience in testing 46 patients for HPP and differential diagnosis by targeted NGS and show that this strategy is efficient and useful. We used an array including ALPL gene, genes of differential diagnosis COL1A1 and COL1A2 that represent 90% of OI cases, SOX9, responsible for CD, and 8 potentially modifier genes of HPP. Seventeen patients were found to carry a mutation in one of these genes. Among them, only 10 out of 15 cases referred for HPP carried a mutation in ALPL and 5 carried a mutation in COL1A1 or COL1A2. Interestingly, three of these patients were adults with fractures and/or low BMD. Our results indicate that HPP and OI may be easily misdiagnosed in the prenatal stage but also in adults with mild symptoms for these diseases.

Hypophosphatasia presenting with pyridoxine-responsive seizures, hypercalcemia, and pseudotumor cerebri: case report.

Hypophosphatasia (HPP) is an inborn error of metabolism characterized by defective bone mineralization caused by a deficiency in alkaline phosphatase (ALP) activity due to mutations in the tissue-nonspecific ALP (TNALP) gene. The clinical expression of the disease is variable. Six forms of HPP are identified according to age at presentation and clinical features. Patients with the infantile form are normal at birth. First symptoms appear within the first 6 months of life. Along with skeletal findings, HPP patients may present with hypercalcemia, seizures, pseudotumor cerebri, and pulmonary insufficiency. Seizures in HPP are refractory to conventional antiepileptic drugs, but are responsive to pyridoxine. Herein, we report a case of HPP who presented with pyridoxine-responsive seizures in the early neonatal period and was found to have hypercalcemia, skeletal demineralization and increased intracranial pressure.

Prenatal diagnosis of familial lethal hypophosphatasia using imaging, blood enzyme levels, chorionic villus sampling and archived fetal tissue.

Hypophosphatasia is an inheritable disorder characterized by defective bone mineralization and a deficiency of tissue-nonspecific alkaline phosphatase (TNSALP) activity. Screening for mutations in the TNSALP gene allows genetic counseling and prenatal diagnosis of the disease in families with severe forms of hypophosphatasia. A 33-year-old, gravida 4, para 3 Japanese woman was referred to Nagoya City University Hospital for prenatal genetic counseling because of two previous occurrences of fetal bone anomalies. The molecular examination showed that the fetus was homozygous for the TNSALP gene mutation c.1559delT, each parent being heterozygous. Genetic counseling was offered and at the next pregnancy, chorionic villus sampling was performed, whereupon genetic analysis confirmed that the fetus did not carry the familial mutation c.1559delT. Postnatal molecular genetic analysis using the cord tissue can provide a diagnosis of lethal hypophosphatasia and prenatal genetic diagnosis of the TNSALP gene allows time for parental counseling and delivery planning.

Hyperphosphatasia with seizures, neurologic deficit, and characteristic facial features: Five new patients with Mabry syndrome.

Persistent hyperphosphatasia associated with developmental delay and seizures was described in a single family by Mabry et al. 1970 (OMIM 239300), but the nosology of this condition has remained uncertain ever since. We report on five new patients (two siblings, one offspring of consanguineous parents, and two sporadic patients) that help delineate this distinctive disorder and provide evidence in favor of autosomal recessive inheritance. Common to all five new patients is facial dysmorphism, namely hypertelorism, a broad nasal bridge and a tented mouth. All patients have some degree of brachytelephalangy but the phalangeal shortening varies in position and degree. In all, there is a persistent elevation of alkaline phosphatase activity without any evidence for active bone or liver disease. The degree of hyperphosphatasia varies considerably ( approximately 1.3-20 times the upper age-adjusted reference limit) between patients, but is relatively constant over time. In the first family described by Mabry et al. 1970, at least one member was found to have intracellular inclusions on biopsy of some but not all tissues. This was confirmed in three of our patients, but the inclusions are not always observed and the intracellular storage material has not been identified.

Orodental phenotype and genotype findings in all subtypes of hypophosphatasia.

BACKGROUND: Hypophosphatasia (HP) is a rare inherited disorder characterized by a wide spectrum of defects in mineralized tissues and caused by deficiency in the tissue non-specific alkaline phosphatase gene (ALPL). The symptoms are highly variable in their clinical expression, and relate to numerous mutations in this gene. The first clinical sign of the disease is often a premature loss of deciduous teeth, mostly in the moderate forms. AIM: The purpose of this study was to document the oral features of HP patients and to relate theses features to the six recognized forms of HP in 5 patients with known genotype and to investigate the genotype-phenotype correlations. METHODS: Clinical and radiographic examinations were carried out. We collected medical and dental history in the kindred and biochemical data. Finally, mutations in the ALPL gene were tested by DNA sequencing in SESEP laboratory. RESULTS: We have for the first time related the known dental anomalies which occur as integral features of HP to the recognized clinical forms of HP. We also pointed out striking dental abnormalities which were never described in association with this rare disease. Accurate genotype-phenotype severity correlations were observed. CONCLUSION: This work allowed us to compare orodental manifestations in all the clinical forms of HP within the patient's sample. According to the severity of the disorder, some dental defects were infrequent, while other were always present. The long term prognosis of the permanent teeth varies from a patient to another. As premature loss of primary teeth is often the first, and sometimes the only visible symptom of the milder forms, the paediatric dentist plays a critical role in the detection and diagnosis of the disease.

Specific ultrasonographic features of perinatal lethal hypophosphatasia.

Prenatal diagnosis of perinatal lethal hypophosphatasia (PL-HPH) by ultrasonography is difficult as PL-HPH must be differentiated from other skeletal dysplasias with short long bones and poor mineralization of the skeleton, such as osteogenesis imperfecta type II and achondrogenesis/hypochondrogenesis. Here we present a case of molecularly confirmed PL-HPH and illustrate specific ultrasonographic findings that help to distinguish PL-HPH from similar conditions.

Chronic multifocal non-bacterial osteomyelitis in hypophosphatasia mimicking malignancy.

BACKGROUND: Hypophosphatasia (HP) is characterized by a genetic defect in the tissue-nonspecific alkaline phosphatase (TNSALP) gene and predominantly an autosomal recessive trait. HP patients suffer from reduced bone mineralization. Biochemically, elevated concentrations of substrates of TNSALP, including pyridoxal-5'-phosphate and inorganic pyrophosphate occur in serum, tissues and urine. The latter has been associated with chronic inflammation and hyperprostaglandinism. CASE PRESENTATION: We report on 2 affected children presenting with multifocal inflammatory bone lesions mimicking malignancy: A 6 years old girl with short stature had been treated with human growth hormone since 6 months. Then she started to complain about a painful swelling of her left cheek. MRI suggested a malignant bone lesion. Bone biopsy, however, revealed chronic inflammation. A bone scan showed a second rib lesion. Since biopsy was sterile, the descriptive diagnosis of chronic non-bacterial osteomyelitis (CNO) was established. The diagnostic tests related to growth failure were repeated and subsequent analyses demonstrated a molecular defect in the TNSALP gene. The second girl (10 years old) complained about back pain after she had fallen from her bike. X rays of her spine revealed compressions of 2 thoracic vertebrae. At first these were considered trauma related, however a bone scan did show an additional lesion in the right 4th rib. A biopsy of this rib revealed a sterile lympho- plasmocytoid osteomyelitis suggesting multifocal CNO. Further analyses did show a decreased TNSALP in leukocytes and elevated pyridoxal phosphate in plasma, suggesting a heterozygous carrier status of HP. CONCLUSION: Chronic bone oedema in adult HP and chronic hyper-prostaglandinism in childhood HP do suggest that in some HP patients bone inflammation is present in conjunction with the metabolic defect. Sterile multifocal osteomyelitis could be demonstrated. Non-steroidal anti-inflammatory treatment achieved complete remission. These cases illustrate chronic inflammation of the bone as a new feature of HP.

Severe cleidocranial dysplasia can mimic hypophosphatasia.

UNLABELLED: Cleidocranial dysplasia (OMIM 119600) is a skeletal dysplasia caused by mutations in the bone/cartilage specific osteoblast transcription factor RUNX2 gene. It is characterised by macrocephaly with persistently open sutures, absent or hypoplastic clavicles, dental anomalies, and delayed ossification of the pubic bones. A few patients have been reported with recurrent fractures or osteoporosis but these are not considered features of the disease. We report a patient with classical findings of cleidocranial dysplasia: markedly hypoplastic clavicles, delayed ossification of the pubic rami, multiple pseudoepiphyses of the metacarpals, and dental anomalies including delayed eruption of permanent dentition and multiple supernumerary teeth. The patient also had radiographic and biochemical features of hypophosphatasia (OMIM 241500, 146300) and was initially diagnosed with this condition. Serum alkaline phosphatase activity has been consistently reduced and specific enzyme substrates, phosphoethanolamine and pyridoxal-5'-phosphate, have been elevated. However, no mutations were found on direct sequencing of the tissue-nonspecific alkaline phosphatase ( TNSALP) gene using a protocol that detects up to 94% of all mutations causing hypophosphatasia. CONCLUSION: We propose that a subset of patients with cleidocranial dysplasia have features of secondary hypophosphatasia due to decreased expression of the tissue-nonspecific alkaline phosphatase gene.