Tissue-nonspecific Alkaline Phosphatase Gene Research Articles

7655 Genetic Mysteries Unveiled: Unusual Mutation Unraveled In Hypophosphatasia

Abstract Disclosure: A. Sharma: None. P. Katikaneni: None. S. Tanveer: None. K. Selk: None. Introduction: Hypophosphatasia (HPP), an inherited dental-osseous metabolic illness, is caused by inactivating mutations in the gene encoding the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP), which leads to a decrease in TNSALP enzymatic activity. Patients with HPP typically experience dental and skeletal mineralization issues as their most common symptoms; musculoskeletal abnormalities, numerous fractures, tiredness, and migraines are less common clinical presentations. Case Report: Our 75-year-old male patient initially presented 1.5 years prior following a spontaneous T12 vertebral fracture. A bone biopsy was performed and did not reveal evidence of malignancy. Testing for SPEP/UPEP was not consistent with myeloma, and thyroid testing was also unrevealing. His alkaline phosphatase level was consistently <40 IU/L since 1999. An evaluation was done for low alkaline phosphatase and secondary reasons for bone loss. This testing did not reveal vitamin D deficiency, parathyroid disease, hypophosphatemia, laboratory evidence of celiac disease, hypogonadism, or hypomagnesemia. A vitamin b6 level was markedly elevated at 119 ng/mL (ref range 2.1-21.7) while not on supplementation. He had noticed multiple fractures as a child, but he had written them off as minor injuries. He could clearly remember breaking his right wrist and clavicle. As an adult, he had rib fractures and a foot fracture. He was unable to recollect losing his primary teeth. Almost all his teeth are crowns or implants despite taking exceptional care of them. He did not have joint hypermobility. Genetic testing revealed the patient was heterozygous for an ALPL gene variant, c.876_882delinsT. It was a variant of undetermined significance at that time. His daughter also was found to have a low alkaline phosphatase and the same genetic test result. She also has experienced dental carries and joint pain. Our patient initiated teriparatide due to the need for more urgent therapy for extensive spinal surgery. He was later transitioned to asfotase alfa therapy and has not had any further fractures at the time of this report. Discussion: Hypophosphatasia is a rare inborn disorder due to mutations in the tissue nonspecific alkaline phosphatase gene ALPL and is characterized by low ALP levels. Affected individuals will also have elevated vitamin b6 levels and urinary phosphoethanolamine (PEA) levels. Early diagnosis of HPP is necessary to prevent bone fracture pain and improve quality of life. Inaccurate assessments can miss diagnoses of parents and other family members and even lead to lost opportunities to treat a patient with a substantial illness burden. This case highlights the mutation c.876_882delinsT as a likely pathologic variant in the ALPL gene. It also supports that patients with HPP can also respond to teriparatide treatment. Presentation: 6/2/2024

Tissue nonspecific alkaline phosphatase deficiency impairs Purkinje cell development and survival in a mouse model of infantile hypophosphatasia.

Loss-of-function mutations in the tissue-nonspecific alkaline phosphatase (TNAP) gene can result in hypophosphatasia (HPP), an inherited multi-systemic metabolic disorder that is well-known for skeletal and dental hypomineralization. However, emerging evidence shows that both adult and pediatric patients with HPP suffer from cognitive deficits, higher measures of depression and anxiety, and impaired sensorimotor skills. The cerebellum plays an important role in sensorimotor coordination, cognition, and emotion. To date, the impact of TNAP mutation on the cerebellar circuitry development and function remains poorly understood. The main objective of this study was to investigate the roles of TNAP in cerebellar development and function, with a particular focus on Purkinje cells, in a mouse model of infantile HPP. Male and female wild type (WT) and TNAP knockout (KO) mice underwent behavioral testing on postnatal day 13-14 and were euthanized after completion of behavioral tests. Cerebellar tissues were harvested for gene expression and immunohistochemistry analyses. We found that TNAP mutation resulted in significantly reduced body weight, shorter body length, and impaired sensorimotor functions in both male and female KO mice. These developmental and behavioral deficits were accompanied by abnormal Purkinje cell morphology and dysregulation of genes that regulates synaptic transmission, cellular growth, proliferation, and death. In conclusion, inactivation of TNAP via gene deletion causes developmental delays, sensorimotor impairment, and Purkinje cell maldevelopment. These results shed light on a new perspective of cerebellar dysfunction in HPP.

A Novel Case of Concomitant PHEX and ALPL Mutation In a Family With Rickets

Abstract Currently, no published cases report concomitant X-linked hypophosphatemia (XLH) and adult hypophosphatasia (HPP). Both diseases share clinical phenotypes that are almost indistinguishable. The correct diagnosis may be missed without a standardized laboratory and genetic testing approach. Pathogenic variants in the phosphate regulating endopeptidases homolog X-linked gene (PHEX) and the tissue-nonspecific alkaline phosphatase gene (ALPL) are genes that cause XLH and HPP, respectively. We describe a concomitant yet undescribed genetic pathogenic variant in a family. A 61-year-old woman was referred by orthopedic surgery for the presence of bilateral leg bowing and short stature during the assessment of knee surgery. The patient had a biochemical workup relevant for low serum phosphorus and 1,25-dihydroxy vitamin D and normal alkaline phosphatase (ALP). Genetic analysis revealed pathogenic variants in PHEX and ALPL. Her 42-year-old daughter shared identical symptoms and genetic variants with her mother. Both patients started conventional treatment for XLH with phosphorus and vitamin D, and the daughter later switched to burosumab-twza. Adult XLH and HPP may have similarities in clinical presentation but differ in some essential laboratory findings. Normal ALP levels helped direct our diagnosis toward XLH. However, the diagnosis was challenging due to the presence of concurrent variants in the genes involved. These variants illustrate the significant heterogeneity of the clinical expression.

Prenatal asfotase alfa-mediated enzyme replacement therapy restores delayed calcification in a severe infantile form of hypophosphatasia model mice

Hypophosphatasia (HPP) is a congenital disorder caused by mutations in the tissue-nonspecific alkaline phosphatase (TNALP) gene. The pathogenesis of HPP varies, ranging from severe cases in which there is total absence of fetal bone calcification, which leads to stillbirth, to relatively mild cases in which the effects are confined to the teeth, such as early loss of the primary teeth. In recent years, the establishment of enzyme supplementation as a treatment method has prolonged survival in patients; however, this approach does not provide sufficient improvement for failed calcification. Furthermore, the effects of enzyme replacement therapy on the jawbone and periodontal tissues have not yet been studied in detail. Therefore, in this study, we investigated the therapeutic effects of enzyme replacement therapy on jawbone hypocalcification in mice. Recombinant TNALP was administered to mothers before birth and newborns immediately after birth, and the effect of treatment was evaluated at 20 days of age. The treated HPP mice had improved mandible (mandibular length and bone quality) and tooth quality (root length of mandibular first molar, formation of cementum), as well as improved periodontal tissue structure (structure of periodontal ligament). Furthermore, prenatal treatment had an additional therapeutic effect on the degree of mandible and enamel calcification. These results suggest that enzyme replacement therapy is effective for the treatment of HPP, specifically in the maxillofacial region (including the teeth and mandible), and that early initiation of treatment may have additional beneficial therapeutic effects.

RF30 | PSAT137 Low Level of Alkaline Phosphatase as a Clue to Noncompliance in Patient with Hypophosphatasia

Abstract Introduction Hypophosphatasia is rare, inherited metabolic disorder caused by mutations in the tissue nonspecific alkaline phosphatase gene. This mutation leads to persistently low level of alkaline phosphatase (ALP) which is the hallmark of the hypophosphatasia but also elevated substrates mostly inorganic pyrophosphate (PPi) and pyridoxal-5-phospate (PLP). Enzyme replacement therapy (ERT) with asfotase alpha presents currently available medical treatment, in which administration of enzyme leads to increase in ALP activity up to several thousand units per liter. Measurement of ALP and other biochemical parameters are crucial during follow-up of treatment with ERT. Clinical case: 42-year-old female with medical history significant for early onset scoliosis secondary to hypophosphatasia diagnosed at age of 6 months presented at endocrinology clinic to establish care. In early childhood patient had 3 nontraumatic femur fractures and ankle fracture at age 25. Patient underwent fixation of bilateral proximal femurs and spinal surgery due to scoliosis. She has been started on weight base treatment with asfotase alpha 6 years ago with 2 mg/kg 3 times per week. During current visit patient continued to experience back pain despite surgery treatment and ongoing treatment with asfotase alpha. Her ALP level in blood was 234 u/l. This ALP level was low considering that patient is on 6 years long treatment and careful history taking revealed that patient is taking smaller dosage of asfotase alpha than recommended. Patient explained that due to side effects of medications: palpitations and nervousness she decreased her dose to 2 times per week which led to inappropriate self-medication and non-compliance. Conclusion Although there are many different causes of low level of ALP like osteogenesis imperfecta, vitamin D intoxication, multiple myeloma, vitamin C deficiency, hypothyroidism, Cushing's syndrome and bisphosphonate therapy recent studies found out that prevalence of mild form of hypophosphatasia can be much higher than expected even 1/508. Due to relative rarity of the disease and lack of the awareness of hypophosphatasia among physicians there are usually delay in the diagnosis and management especially in mild forms. Measurement of ALP activity is crucial for diagnosis of hypophosphatasia but is also essential minimum for monitoring of treatment. Relatively low level of ALP while on treatment with asfotase alpha as in our case should prompt for assessment of medication compliance or treatment failure. Further assessment with detailed information about medication injections, dosage, and frequency as well as measurements of biochemical parameters, radiography, pain assessment and mobility are needed in follow up of adults with hypophosphatasia. More awareness and education on diagnosis and follow up of patients with hypophosphatasia are needed so patients can be effectively treated with available enzyme replacement therapy. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m., Monday, June 13, 2022 12:54 p.m. - 12:59 p.m.

Vitamin B-related Gene Polymorphisms and Cardiovascular Disease.

Hyperhomocysteinemia is an independent risk factor for atherosclerosis, even in early childhood. A mutation in genes that code homocysteine metabolism enzymes or deficiency of specific vitamin cofactors may cause hyperhomocysteinemia. Vitamin B complex has been correlated with serum homocysteine levels. Any abnormality in its metabolism or nutritional deficiency may lead to hyperhomocysteinemia. Both vitamin B complex and homocysteine levels are partly genetically determined. Specifically, the most studied polymorphism is 677T-C in exon 5 of the 5,10- methylenetetrahydrofolate reductase (MTHFR) gene, which plays an important role in folate's metabolism. This polymorphism has been shown to be correlated with hypertension and cardiovascular disease. Polymorphisms in methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1-like (MTHFD1L) gene have also been correlated with increased risk for coronary artery disease. Other common serious polymorphisms regard the area with high linkage disequilibrium, including the neuroblastoma breakpoint family, NBPF3 gene, and ~ 12-50 kb upstream of the tissue nonspecific alkaline phosphatase gene. Finally, the polymorphisms which have been mostly associated with vitamin B12 concentration are the rs11254363 polymorphism at intron 52 of the intrinsic factor vitamin B12 receptor of the CUBN and the rs526934 polymorphism at intron 8 of transcobalamin I. To sum up, several polymorphisms have already been associated with vitamin B complexes and therefore homocysteine level, highlighting the complex nature of vitamin B genetics.

Polymorphic variants of alkaline phosphatase gene correlate with clinical signs of adult hypophosphatasia?

SummaryWe analyzed polymorphism of the ALPL gene in patients with low serum levels of tissue-nonspecific alkaline phosphatase (TNAP). The presence of three or more of the less frequent alleles of ALPL polymorphisms was associated with significantly lower TNAP serum level and higher frequencies of metatarsal fractures, which may help confirm a clinical suspicion of adult hypophosphatasia.IntroductionAlkaline phosphatases (ALPs) are membrane-bound enzymes that hydrolyze monophosphate esters at a high pH (pH 8–10). Inorganic pyrophosphate, pyridoxal 5-phosphate, the activated form of vitamin B6 (PLP), and phosphoethanolamine (PEA), are natural substrates of ALPs. Hypophosphatasia (HPP, OMIM 146300, 241500, 241510) is a heterogeneous rare metabolic bone disease caused by loss-of-function mutations in the tissue-nonspecific alkaline phosphatase gene (ALPL; MIM 171760) with a deficiency of TNAP. Clinical presentation of HPP in adults demonstrated a wide range of manifestations, many of which are nonspecific. In the present study, we screened the polymorphic genetic variants of ALPL in 56 subjects presenting low serum levels of TNAP and/or other clinical signs of adult HPP in order to evaluate a possible role of polymorphic variants in the diagnosis and management of HPP in adults.MethodsGenomic DNA was extracted from peripheral blood and ALPL gene was sequenced by PCR-based Sanger technique.ResultsFourteen different polymorphic variants were found in the study population. A lower serum level of TNAP and higher frequencies of metatarsal fractures were observed in patients bearing three or more of the minor frequency alleles (MFAs) of the ALPL polymorphic variants. The presence of some MFAs, mostly as a contemporary presence of three or more of them, was found to be mainly represented in patients having both a significantly lower level of TNAP and a higher level of vitamin B6.ConclusionThe genetic analysis and presence of some polymorphic variants may be an instrument to confirm clinical and biochemical data, consider adult HPP, and help clinicians be cautious in the administration of anti-reabsorption drugs.

A Rare Presentation of Non-Traumatic Recurring Bone Fractures in Adult Patient With Juvenile Onset Hypophosphatasia

Background: Hypophosphatasia is a rare metabolic bone disease caused by mutations in the tissue-nonspecific alkaline (TNSALP) phosphatase gene. The disease is typically diagnosed in children but may be seen in adults with premature loss of adult teeth, poor bone healing, recurrent bone fractures, and limited mobility due to chronic pain. Clinical case: A 48-year-old female with Juvenile Onset hypophosphatasia and diabetes mellitus type 2 who presented to the endocrine clinic for diabetes mellitus. She had a telemedicine visit because she was not able to come to the office due to her recent rib fracture and pain. She had multiple fractures since she was 16-years-old with multiple fractures in her toes. She later developed a femur fracture when 20 years old and second at 28-years-old in the other femur. She was tested for juvenile hypophosphatasia and the diagnosis was confirmed. She received no treatment at that time. She subsequently had recurrent non-traumatic rib fractures that interfered with daily life. She is unable to exercise due to her frequent fractures and consequently having poor diabetic control. She has family history of brother with femur fracture. Labs showed decreased alkaline phosphatase (<5 normal: 38–126 U/L), elevated vitamin B6 levels (2296.2: normal 20–125 nmol/L) normal vitamin D (33: normal 30–100 ng/mL) and calcium levels. Chest x-ray showed multiple rib fractures with different healing stages. Dexa scan was also done and was normal to age with lowest Z-score being 8.2. Patient was started on Asfotase Alfa (an enzyme replacement therapy that contains the active site of TNSALP). Conclusion: This case illustrates the debilitating effects of hypophosphatasia and importance of recognizing previously untreated adult patients with hypophosphatasia. Many patients have very late diagnosis and suffer severe complications in their adulthood. Although this disease is rare, recognizing the symptoms of juvenile onset hypophosphatasia can help initiate early treatment to decrease these complications. Hypophosphatasia should be considered as a differential diagnosis in adults with multiple fractures and decreased alkaline phosphatase.

One patient with perinatal hypophosphatasia due to mutations in the tissue-nonspecific alkaline phosphatase gene

Objective To explore the clinical and genetic characteristics of a Chinese baby with perinatal hypophosphatasia (HPP) and his parents for better understanding of the disease. Methods The clinical data of the patient with HPP was carefully collected. The laboratory and radiographic examination data were taken for this baby patient. Sequencing for all the twelve tissue-nonspecific alkaline phosphatase(ALPL) exons and the flanking exon-intron junctions were performed in the proband and his parents with their genomic DNA from peripheral blood. Results The blood level of alkaline phosphatase was decreased in this patient while serum calcium level was increased. His bone revealed chondrodysplasia-like change. Compound heterozygous mutations were found in the proband, with c. 346G>A(p.A116T) in exon 5 and c. 1171C>T(p.R391C) in exon 10. His father and mother were without clinical manifestation while respectively carried c. 346G>A(p.A116T and c. 1171C>T(p.R391C) missense mutations, suggesting an autosomal recessive inheritance in this family. Conclusion Perinatal HPP has a high mortality rate. Skeletal deformities, hypercalcemia, and low level of ALP are important in the differential diagnosis of perinatal HPP. Key words: Mutation; Hypophosphatasia; Alkaline phosphatase gene

Hypophosphatasia: Biological and Clinical Aspects, Avenues for Therapy.

Hypophosphatasia (HPP) is a rare inherited systemic metabolic disease caused by mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene. TNSALP is expressed in the liver, kidney and bone, and its substrates include TNSALP inorganic pyrophosphate, pyridoxal-5'-phosphate (PLP)/vitamin B6 and phosphoethanolamine (PEA). Autosomal recessive and dominant forms of the disease result in a range of clinical entities. Major hallmarks are low alkaline phosphatase (ALP) and elevated PLP and PEA levels. Very severe infantile forms of HPP cause premature death as a result of respiratory insufficiency and also present with hypo-mineralisation leading to deformed limbs with, in some cases, the near-absence of bones and skull altogether. Respiratory failure, rib fractures and seizures due to vitamin B6 deficiency are indicative of a poor prognosis. Craniosynostosis is frequent. HPP leads to an unusual presentation of rickets with high levels of calcium and phosphorus, resulting in hypercalciuria, nephrocalcinosis and low ALP levels. Hypercalcaemic crisis, failure to thrive and growth retardation are concerns in infants. Fractures are common in both infantile and adult forms of the disease, concomitantly occurring with unexplained chronic pain and fatigue. Dental clinical presentations, which include the premature loss of teeth, are also commonly found in HPP and specifically manifest as odontohypophosphatasia. A novel enzyme therapy for human HPP, asfotase alfa, which is specifically targeted to mineralised tissues, has been developed in the past decades. While this treatment seems very promising, especially for infantile HPP, many questions regarding its long-term effects, the management of treatment, and any potential secondary adverse effects remain unresolved.

Adult hypophosphatasia with compound heterozygous p.Phe327Leu missense and c.1559delT frameshift mutations in tissue-nonspecific alkaline phosphatase gene: a case report

BackgroundHypophosphatasia is an inherited bone disease characterized by low alkaline phosphatase activity encoded by ALPL. Clinically, hypophosphatasia can be categorized as perinatal, infantile, childhood, and adult forms, as well as odonto-hypophosphatasia, according to the age at first sign or dental manifestations. Adult hypophosphatasia typically presents in middle-aged patients who appear to be in good health in early adulthood and manifests as painful feet caused by recurrent, slow-healing stress fractures of the lower limb. Because the symptoms of adult hypophosphatasia vary and are common, many patients with hypophosphatasia might be not diagnosed accurately and thus may receive inappropriate treatment.Case presentationWe report a case of a 35-year-old Japanese woman with low serum alkaline phosphatase detected at a routine medical checkup. She had mild muscle/bone pain but no history of rickets, fractures, or dental problems. Measurement of bone mineral density of the lumbar spine and the femoral neck revealed osteopenia below the expected range for age in a young adult. Abdominal ultrasonography revealed numerous microcalcifications in both kidneys. Analysis of amino acids in urine revealed that phosphoethanolamine was elevated. Low serum alkaline phosphatase activity, elevation of phosphoethanolamine, and low bone mineral density supported the diagnosis of hypophosphatasia. ALPL mutation analysis revealed two mutations: p.Phe327Leu and c.1559delT. These genetic abnormalities were previously reported in perinatal, infantile, and childhood but not adult hypophosphatasia. On the basis of the clinical presentation, laboratory and imaging findings, and genetic analyses, the patient was definitively diagnosed with adult hypophosphatasia. To the best of our knowledge, this is the first case report of adult hypophosphatasia with the compound heterozygous mutations p.Phe327Leu and c.1559delT.ConclusionsAlthough the risk of bone fracture was high in this case, treatment approaches differ between osteoporosis and hypophosphatasia. Because adult hypophosphatasia diagnosis is often difficult because of their varied symptoms, hypophosphatasia should be considered in the differential diagnosis of low serum alkaline phosphatase. Early diagnosis is important so that appropriate treatment can be initiated.

MON-493 A Unique Case of Severe Osteogenesis Imperfecta Due to a Novel Heterozygous Mutation in COL1A1 Gene with Overlapping Hypophosphatasia Phenotype

Background: Osteogenesis imperfecta (OI) is a rare heterogeneous bone fragility disease due to collagen defect most often caused by autosomal dominant (AD) COL1A1 or COL1A2 gene mutations. Severe forms of Hypophosphatasia (HPP) may phenotypically resemble OI, is a distinct disorder of hypomineralization caused by the mutations in the tissue-nonspecific alkaline phosphatase (ALPL) gene. Here in, we present the first case of a neonate with severe perinatal OI caused by a novel AD mutation in COL1A1 gene with HPP phenotype treated with asfotase alfa. Case: A 3-day-old full-term boy born by cesarean section presented with multiple fractures & dislocated hips at birth. On physical exam, he was found to have respiratory distress ultimately requiring intubation, hypoplastic rib-cage, blue sclerae, mesomelia, rhizomelia, left & right dislocated hips. Skeletal survey showed multiple fractures of long bones and ribs of varying ages, characteristic of OI. In addition, he was noted to have generalized hypomineralization, hypoplastic ribs, and some metaphyseal flaring. Interestingly, biochemical evaluation revealed hypercalcemia (corrected calcium of 11.7 mg/dL), low alkaline phosphatase (ALP) 58(80-270 U/L), slightly elevated pyridoxal 5” phosphate 52 (5-50 mg/dL) & high urine phosphoethanolamine 285(83-222 mcmol/g Cr). Due to clinical, radiographic, and biochemical findings concerning for HPP, the patient was started on asfotase alfa. The genetic testing showed a novel likely pathogenic heterozygous variant Chr17: c.1201G>C (p.Gly401Arg) in the COL1A1 gene consistent with AD OI. The ALPL gene sequencing revealed no variants in the coding region with 100% coverage of the region. After 7 wks of treatment, x-rays showed two new long bone fractures, but interval osseous remodeling & healing of the previous fractures with an overall improvement in the tubulation of the long bones. Conclusion: To our knowledge, this is the first report describing low ALP activity with phenotypic features of HPP in a neonate with severe perinatal OI due to a novel AD COL1A1 mutation. The low ALP could be due to osteoblasts dysfunction rather than ALPL gene causing enzyme deficiency & it highlights a potentially important role of osteoblasts in the pathogenesis of OI leading to reduced ALP activity and HPP phenotype. Interestingly, patients with lethal type II OI exhibit hypoplastic ribs and generalized hypomineralization; and, osteoblastic dysfunction has been suggested. It is tempting to speculate that low ALP activity may potentially have an additive effect on the severity and possibly lethality of certain most severe forms of OI contributing to potentially a new phenotype of this heterogeneous disorder. Long-term follow-up is needed to see if the patient will benefit from asfotase alfa treatment. In addition, further studies should be performed to truly understand the pathogenesis of the disease in this patient.

Interference of Asfotase Alfa in Immunoassays Employing Alkaline Phosphatase Technology.

Asfotase alfa (STRENSIQ®, Alexion Pharmaceuticals, Inc.) is the only approved treatment for patients with pediatric-onset hypophosphatasia, a disease caused by a mutation in the tissue-nonspecific alkaline phosphatase (TNSALP) gene. ALP is often used as signaling system in routine immunoassays. Because asfotase alfa contains the active site of the full ALP enzyme, it can catalyze the substrate as the antibody-conjugated ALP would within an assay. Therefore, its presence in a treated patient's sample may generate false positive or false negative results. We investigated whether the presence of asfotase alfa within a sample induced interference in immunoassays that utilize ALP or alternative detection systems. Asfotase alfa was added to samples at concentrations from 0.08-5 µg/mL and analysed on various immunoassays following manufacturer's instructions. Asfotase alfa was detected in all ALP assays but ALKP1 (RayBiotech). We observed no changes in normetanephrine and noradrenaline (IBL) at any asfotase alfa concentration. However, asfotase alfa notably interfered in an oxytocin (ENZO) assay in nonextracted samples. Extraction using a C18 column eliminated the interference. No interference was observed on automated analyzers using alternative detection system (COBAS fT4 and TSH; Advia Centaur FSH, fT4; Architect LH; FSH). Immulite 2000 fT4, TSH, testosterone and hCG (ALP-based) showed no interference. However, the presence of asfotase alfa resulted in a dose-dependent increase of Troponin I signal. The presence of asfotase alfa must be taken into consideration when analyzing blood samples in treated patients to avoid any risk of misinterpretation of false positive/negative results. It is essential that assays be tested for this possible interference.

A homozygous missense variant in the alkaline phosphatase gene ALPL is associated with a severe form of canine hypophosphatasia

Inherited skeletal disorders affect both humans and animals. In the current study, we have performed series of clinical, pathological and genetic examinations to characterize a previously unreported skeletal disease in the Karelian Bear Dog (KBD) breed. The disease was recognized in seven KBD puppies with a variable presentation of skeletal hypomineralization, growth retardation, seizures and movement difficulties. Exome sequencing of one affected dog revealed a homozygous missense variant (c.1301T > G; p.V434G) in the tissue non-specific alkaline phosphatase gene, ALPL. The identified recessive variant showed full segregation with the disease in a cohort of 509 KBDs with a carrier frequency of 0.17 and was absent from 303 dogs from control breeds. In humans, recessive and dominant ALPL mutations cause hypophosphatasia (HPP), a metabolic bone disease with highly heterogeneous clinical manifestations, ranging from lethal perinatal hypomineralization to a relatively mild dental disease. Our study reports the first naturally occurring HPP in animals, resembling the human infantile form. The canine HPP model may serve as a preclinical model while a genetic test will assist in breeding programs.

Genetically Engineering a Sheep Model of Hypophosphatasia

Hypophosphatasia (HPP) is a rare inherited disorder that affects the development of bones and teeth. The disease is caused by mutations in the tissue‐nonspecific alkaline phosphatase (TNSALP) gene (ALPL) and accompanied by a highly variable clinical presentation. Although HPP patient studies have advanced our understanding of HPP, as well as documented disease severity, the rarity of the disease combined with a lack of sufficient animal models has significantly delayed mechanistic understanding and therapeutic development. Similar to other human disease models, current HPP models have been engineered virtually exclusively in rodents – specifically mice harboring null or loss‐of‐function mutations. Although useful for modeling some features of HPP, these murine models do not faithfully represent the broad spectrum of human HPP clinical bone, muscle and tooth (odonto‐HPP) phenotypes. However, in sheep both Haversian bone remodeling and tooth development are analogous to humans with sheep TNSALP amino acid sequence sharing 89% identity with human. Thus, our objective was to generate a sheep model of HPP and we posit that ALPL mutation‐specific replacements in the sheep genome using CRISPR/Cas9 will produce a model that accurately phenocopies the bone and tooth pathophysiology of HPP. To genetically engineer a sheep model of HPP, a total of 52 in vitro genetically manipulated embryos were generated targeting the Exon 10 c.1077G&gt;A mutation using CRISPR/Cas9. Three embryos were implanted per recipient ewe and implantations were performed in a total of 17 recipient ewes. Nine ewe pregnancies were confirmed by the measurement of Pregnancy‐Associated Protein on post‐implantation day 35 and further validated by ultrasound. From 9 live newborn lambs, four were heterozygous for our specific point mutation (4/6), one compound heterozygous (1/6) and one homozygous (1/6) for a total mutation efficiency rate of 66.6% (6/9). Similar to HPP patients with the same mutation, mutant sheep have variable clinical expression accompanied with decreased bone formation and mineralization in tails observed by DXA as well as significantly and appropriately decreased serum alkaline phosphatase activity. Additionally, an apparent “shell tooth” phenotype is observed in mutant lambs, further suggesting a mutant exon 10 genotype/phenotype that more completely recapitulates human skeletal and odonto‐HPP.Support or Funding Information1. William Townsend Porter Pre‐doctoral Fellowship2. TAMU/Texas Heart Institute Center for Cell and Organ Biotechnology Innovation Kitchen GrantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

ATP catabolism by tissue nonspecific alkaline phosphatase contributes to development of ARDS in influenza-infected mice.

Influenza A viruses are highly contagious respiratory pathogens that are responsible for significant morbidity and mortality worldwide on an annual basis. We have shown previously that influenza infection of mice leads to increased ATP and adenosine accumulation in the airway lumen. Moreover, we demonstrated that A1-adenosine receptor activation contributes significantly to influenza-induced acute respiratory distress syndrome (ARDS). However, we found that development of ARDS in influenza-infected mice does not require catabolism of ATP to adenosine by ecto-5'-nucleotidase (CD73). Hence, we hypothesized that increased adenosine generation in response to infection is mediated by tissue nonspecific alkaline phosphatase (TNAP), which is a low-affinity, high-capacity enzyme that catabolizes nucleotides in a nonspecific manner. In the current study, we found that whole lung and BALF TNAP expression and alkaline phosphatase enzymatic activity increased as early as 2 days postinfection (dpi) of C57BL/6 mice with 10,000 pfu/mouse of influenza A/WSN/33 (H1N1). Treatment at 2 and 4 dpi with a highly specific quinolinyl-benzenesulfonamide TNAP inhibitor (TNAPi) significantly reduced whole lung alkaline phosphatase activity at 6 dpi but did not alter TNAP gene or protein expression. TNAPi treatment attenuated hypoxemia, lung dysfunction, histopathology, and pulmonary edema at 6 dpi without impacting viral replication or BALF adenosine. Treatment also improved epithelial barrier function and attenuated cellular and humoral immune responses to influenza infection. These data indicate that TNAP inhibition can attenuate influenza-induced ARDS by reducing inflammation and fluid accumulation within the lung. They also further emphasize the importance of adenosine generation for development of ARDS in influenza-infected mice.

Specific knock-down of tissue non-specific alkaline phosphatase mRNA levels inhibits intracellular lipid accumulation in 3T3-L1 and HepG2 cells.

The use of non-specific inhibitors of tissue non-specific alkaline phosphatase (TNSALP) in pre-adipocytes blocks intracellular lipid accumulation. TNSALP is also expressed in hepatocytes, which are known to accumulate lipid in a similar manner to pre-adipocytes. The purpose of this study was to use specific silencing of TNSALP mRNA, using short interfering (si) RNA, to investigate the role of TNSALP in intracellular lipid accumulation in 3T3-L1 and HepG2 cells. Cellular activity of TNSALP was measured using an automated colorimetric assay, and intracellular lipid accumulation was determined using the lipid-specific dye, Oil Red O. Cells were transfected with siRNA directed against TNSALP mRNA, and expression of the TNSALP gene was determined at selected time points postinduction of lipid droplet formation. Expression of the TNSALP gene was inhibited by a maximum of 88±1.9% (P<0.005 vs. control) 11days after initiation of lipid droplet formation in the 3T3-L1 cells and 80±8.9% (P<0.05 vs. control) after 4days in the HepG2 cells. This led to significant inhibition of both TNSALP activity and intracellular lipid accumulation in both cell lines. These data demonstrates that TNSALP plays an important role in the control of lipid droplet formation in both pre-adipocyte and hepatocyte cell lines.

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 PNMR 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 and clinical analysis of ALPL in a cohort of patients with suspicion of Hypophosphatasia.

Hypophosphatasia (HPP) is a rare autosomal dominant or recessive metabolic disorder caused by mutations in the tissue nonspecific alkaline phosphatase gene (ALPL). To date, over 300 different mutations in ALPL have been identified. Disease severity is widely variable with severe forms usually manifesting during perinatal and/or infantile periods while mild forms are sometimes only diagnosed in adulthood or remain undiagnosed. Common clinical features of HPP are defects in bone and tooth mineralization along with the biochemical hallmark of decreased serum alkaline phosphatase activity. The incidence of severe HPP is approximately 1 in 300,000 in Europe and 1 in 100,000 in Canada. We present the clinical and molecular findings of 83 probands and 28 family members, referred for genetic analysis due to a clinical and biochemical suspicion of HPP. Patient referrals included those with isolated low alkaline phosphatase levels and without any additional clinical features, to those with a severe skeletal dysplasia. Thirty-six (43.3%) probands were found to have pathogenic ALPL mutations. Eleven previously unreported mutations were identified, thus adding to the ever increasing list of ALPL mutations. Seven of these eleven were inherited in an autosomal dominant manner while the remaining four were observed in the homozygous state. Thus, this study includes a large number of well-characterized patients with hypophosphatasemia which has permitted us to study the genotype:phenotype correlation. Accurate diagnosis of patients with a clinical suspicion of HPP is crucial as not only is the disease life-threatening but the patients may be offered bone targeted enzymatic replacement therapy. © 2017 Wiley Periodicals, Inc.

Hypophosphatasia: Review of Bone Mineral Metabolism, Pathophysiology, Clinical Presentation, Diagnosis, and Treatment

Hypophosphatasia (HPP) is a rare, inherited form of rickets or osteomalacia due to reduced activity of tissue non-specific alkaline phosphatase (TNSALP) caused by a loss of function mutation in the TNSALP gene marked by a low serum alkaline phosphatase. The ratio of PPi to Pi is crucial in the mineralization process. This process is regulated by the interaction of three phosphatases present in matrix vesicles: the mineralization promotors TNSALP and phosphatase orphan 1 (PHOSPHO1) and the mineralization inhibitor nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1). HPP is seen worldwide affecting all races. The broad-ranging clinical severity of HPP is correlated to the pattern of inheritance and degree of TNSALP activity, with lower levels of activity seen in the more severe autosomal recessive forms. HPP is marked by compromise of phase 2 hydroxyapatite crystal formation beyond the matrix vesicle resulting in skeletal hypomineralization, spontaneous fractures, tooth loss, and chondrocalcinosis. The major forms in declining order of severity include the following: perinatal, infantile, childhood, and adult HPP in addition to OdontoHPP and benign prenatal HPP. Biochemistry typically reveals a low serum alkaline phosphatase and elevated alkaline substrates such as pyridoxal-5′-phosphate (PLP) and phosphoethanolamine (PEA). Asfotase alfa, a recent novel enzyme replacement therapy for the treatment of HPP, has transformed this once lethal disease to one that is less severe.