ALPL Mutations 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

7077 A Rare Case Of Hypophosphatasia Presenting With Osteoporosis In A Young Female Patient- Diagnostic Delay In Hypophosphatasia

Abstract Disclosure: R. Abdelmasih: None. I. Ali: None. Introduction: Hypophosphatasia is a rare hereditary cause of secondary osteoporosis that affects 1/100.000 individuals. The rate of osteoporosis due to hypophosphatasia is probably underestimated as it likely goes undiagnosed in heterogeneous or milder cases. We present a case of Hypophosphatasia that presented initially with osteoporosis and went misdiagnosed for years with intermittent treatment of Bisphosphonate, luckily with no atypical fractures that prompted different course of management for osteoporosis. Case Presentation: A 44-year-old female with a history of rheumatoid arthritis and osteoporosis diagnosed 7 years prior and treated with intermittent bisphosphonate which was discontinued due to side effects including muscle aches and bone pains, presented to our endocrinology clinic to establish care for osteoporosis. Patient denied prior fractures. She reported jaw pain that is being evaluated by otolaryngology and a dentist. Family history is negative. DEXA scan showed Z scores of -1.1, -1.8, and -2.2 of the lumbar spine, femoral neck, and total femur respectively. Laboratory workup showed consistently low alkaline phosphatase (ALP) 25, 20, <20 U/L (ref range 34-123), elevated Vitamin B6 of 167 nmol/L (ref range 20-125), normal parathormone, calcium, phosphate, Vit D, magnesium, and liver enzymes. Genetic testing was sent for Tissue non-specific alkaline phosphatase (TNALP). Hypophosphatasia was diagnosed based on clinical and laboratory findings, patient was started on Asfotase alfa. Clinical course is to be followed. Discussion: Hypophosphatasia is a rare hereditary disorder due to mutation of (TNALP). In normal induvial, TNALP breaks down pyrophosphate to phosphate. It also plays a role in transferring Vit B6 into the tissues. Hence, clinical presentation due to impaired calcium-phosphate hemostasis is broad including musculoskeletal pain, pseudofractues, dental abscess, and neurological symptoms (due to accumulation of Vit B6). The clinical picture varies depending on the heterogenicity of ALPL mutation. Genetic testing is not mandated to establish diagnosis, clinical presentation and laboratory testing including low ALP and elevated Vit B6 are sufficient. However, genetic testing and counseling are recommended for the patient’s sake and their family members. Antiresorptive bone therapy is contraindicated due to a further decrease of bone turnover with reported cases of increased risk of atypical femur fracture, Bone density is reported to improve with enzyme replacement therapy with bone-targeting recombinant alkaline phosphatase (Asfotase Alpha), if further treatment is needed, anabolic treatment is preferred. We present this case to shed light on such a rare disease to decrease rates of underdiagnosis or delayed diagnosis in young patients presenting with osteoporosis for prompt treatment and improved quality of life of these patients. Presentation: 6/2/2024

Idiopathic juvenile osteoporosis-a polygenic disorder?

Idiopathic juvenile osteoporosis (IJO) is a rare condition presenting with vertebral and metaphyseal fractures that affects otherwise healthy prepubertal children. Bone mineral density (BMD) measurements are very low. The primary problem appears to be deficient bone formation, with a failure to accrue bone normally during growth. The onset in childhood suggests IJO is a genetic disorder, and a number of reports indicate that some children carry heterozygous pathogenic variants in genes known to be associated with defective osteoblast function and low bone mass, most commonly LRP5 or PLS3. However, a positive family history is unusual in IJO, suggesting the genetic background can be complex. We describe a young man with classical IJO who was investigated with a bone fragility gene panel and whole genome sequencing. The proband was found to carry four variants in three different genes potentially affecting osteoblast function. From his mother he had inherited mutations in ALPL (p.Asn417Ser) and LRP5 (p.Arg1036Gln), and from his father mutations in LRP5 (p.Asp1551Alsfs*13) and activating transcription factor 4 (ATF4) (p.Leu306Ile). His sister had also inherited the LRP5 (p.Asp1551Alsfs*13) from her father, but not the ATF4 mutation. Their spinal BMD z-scores differed substantially (sister -1.6, father -3.2) pointing to the potential importance of the ATF4 mutation. Activating transcription factor 4 acts downstream from RUNX2 and osterix and plays an important role in osteoblast differentiation and function. This case, together with others recently published, supports the view that IJO can result from clustering of mutations in genes related to osteoblast development and function. Novel genes in these pathways may be involved. Our case also emphasizes the value of detailed study of other family members. After a bone biopsy had excluded a mineralization defect due to hypophosphatasia, the proband was treated with zoledronate infusions with good clinical effect.

ALPL regulates pro-angiogenic capacity of mesenchymal stem cells through ATP-P2X7 axis controlled exosomes secretion

BackgroundEarly-onset bone dysplasia is a common manifestation of hypophosphatasia (HPP), an autosomal inherited disease caused by ALPL mutation. ALPL ablation induces prototypical premature bone ageing characteristics, resulting in impaired osteogenic differentiation capacity of human bone marrow mesenchymal stem cells (hBMMSCs). As angiogenesis is tightly coupled with osteogenesis, it also plays a necessary role in sustaining bone homeostasis. We have previously observed a decrease in expression of angiogenesis marker gene CD31 in the metaphysis of long bone in Alpl+/− mice. However, the role of ALPL in regulation of angiogenesis in bone has remained largely unknown.MethodsExosomes derived from Normal and HPP hBMMSCs were isolated and identified by ultracentrifugation, transmission electron microscopy, and nanoparticle size measurement. The effects of ALPL on the angiogenic capacity of hBMMSCs from HPP patients were assessed by immunofluorescence, tube formation, wound healing and migration assay. exo-ELISA and Western Blot were used to evaluate the exosomes secretion of hBMMSCs from HPP, and the protein expression of VEGF, PDGFBB, Angiostatin and Endostatin in exosomes respectively.ResultsWe verified that ALPL ablation resulted in impaired pro-angiogenic capacity of hBMMSCs, accounting for reduced migration and tube formation of human umbilical vein endothelial cells, as the quantities and proteins composition of exosomes varied with ALPL expression. Mechanistically, loss of function of ALPL enhanced ATP release. Additional ATP, in turn, led to markedly elevated level of ATP receptor P2X7, which consequently promoted exosomes secretion, resulting in a decreased capacity to promote angiogenesis. Conversely, inhibition of P2X7 increased the angiogenic induction capacity by preventing excessive release of anti-angiogenic exosomes in ALPL deficient-hBMMSCs.ConclusionThe ALPL–ATP axis regulates the pro-angiogenic ability of hBMMSCs by controlling exosomes secretion through the P2X7 receptor. Thus, P2X7 may be proved as an effective therapeutic target for accelerating neovascularization in ALPL–deficient bone defects.

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.

Clinical and molecular description of the first Italian cohort of 33 subjects with hypophosphatasia.

Hypophosphatasia (HPP) is a rare genetic disease caused by inactivating variants of the ALPL gene. Few data are available on the clinical presentation in Italy and/or on Italian HPP surveys. There were 30 suspected HPP patients recruited from different Italian tertiary cares. Biological samples and related clinical, biochemical, and anamnestic data were collected and the ALPL gene sequenced. Search for large genomic deletions at the ALPL locus (1p36) was done. Phylogenetic conservation and modeling were applied to infer the effect of the variants on the protein structure. There were 21 ALPL variants and one large genomic deletion found in 20 out of 30 patients. Unexpectedly, NGS-driven differential diagnosis allowed uncovering three hidden additional HPP cases, for a total of 33 HPP subjects. Eight out of 24 coding variants were novel and classified as "pathogenic", "likely pathogenic", and "variants of uncertain significance". Bioinformatic analysis confirmed that all the variants strongly destabilize the homodimer structure. There were 10 cases with low ALP and high VitB6 that resulted negative to genetic testing, whereas two positive cases have an unexpected normal ALP value. No association was evident with other biochemical/clinical parameters. We present the survey of HPP Italian patients with the highest ALPL mutation rate so far reported and confirm the complexity of a prompt recognition of the syndrome, mostly for HPP in adults. Low ALP and high VitB6 values are mandatory for the genetic screening, this latter remaining the gold standard not only to confirm the clinical diagnosis but also to make differential diagnosis, to identify carriers, to avoid likely dangerous therapy in unrecognized cases.

Clinical and Genetic Characteristics of Hypophosphatasia in Chinese Adults.

Hypophosphatasia (HPP) is an inherited disease caused by ALPL mutation, resulting in decreased alkaline phosphatase (ALP) activity and damage to bone and tooth mineralization. The clinical symptoms of adult HPP are variable, making diagnosis challenging. This study aims to clarify the clinical and genetic characteristics of HPP in Chinese adults. There were 19 patients, including 1 with childhood-onset and 18 with adult-onset HPP. The median age was 62 (32-74) years and 16 female patients were involved. Common symptoms included musculoskeletal symptoms (12/19), dental problems (8/19), fractures (7/19), and fatigue (6/19). Nine patients (47.4%) were misdiagnosed with osteoporosis and six received anti-resorptive treatment. The average serum ALP level was 29.1 (14-53) U/L and 94.7% (18/19) of patients had ALP levels below 40 U/L. Genetic analysis found 14 ALPL mutations, including three novel mutations-c.511C>G (p.His171Ala), c.782C>A (p.Pro261Gln), and 1399A>G (p.Met467Val). The symptoms of two patients with compound heterozygous mutations were more severe than those with heterozygous mutations. Our study summarized the clinical characteristics of adult HPP patients in the Chinese population, expanded the spectrum of pathogenic mutations, and deepened clinicians' understanding of this neglected disease.

Identifying adult hypophosphatasia in the rheumatology unit

BackgroundThe most frequent manifestation in adult hypophosphatasia (HPP) is musculoskeletal pain. The unspecific nature of its clinical presentation may prevent correct diagnosis. The aim of the study was to assess the prevalence of ALPL mutations in adult patients treated in rheumatological outpatient facilities with evident musculoskeletal symptoms typical for HPP.MethodsOver a period of 10 years 9,522 patients were screened in the rheumatology outpatient clinic of the Hanusch hospital Vienna. Serum ALP levels ≤ 40 U/L were found in 524 patients. After screening for secondary causes, 73 patients were invited for clinical evaluation. Genetic testing was performed in 23 patients with suspected HPP. Logistic regression models with Firth penalisation were used to estimate the unadjusted and BMI-adjusted association of each clinical factor with HPP.ResultsMutations in the ALPL gene were observed in 57% of genetically screened patients. Arthralgia, fractures, and pain were the leading symptoms in individuals with ALPL mutation. Chondrocalcinosis (OR 29.12; 95% CI 2.02–1593.52) and dental disease (OR 8.33; 95% CI 0.93–143.40) were associated with ALPL mutation, independent of BMI. Onset of symptoms in patients with ALPL mutation was at 35.1 (14.3) years, with a mean duration from symptoms to diagnosis of 14.4 (8.1) years. Bone mineral density (BMD) and trabecular bone score (TBS) as well as bone turnover markers were not indicative for HPP or ALPL mutation.ConclusionHPP can mimic rheumatologic diseases. Thus, HPP should be considered as a possible diagnosis in adult patients presenting with musculoskeletal pain of unknown origin in rheumatology outpatient clinics. In patients with persistently low ALP serum levels and unclear musculoskeletal pain, HPP as the underlying cause has to be considered.

Urine phosphoethanolamine is a specific biomarker for hypophosphatasia in adults.

We investigated the utility of urine phosphoethanolamine (PEA) as a marker to aid in diagnosing and/or confirming hypophosphatasia (HPP) in adults and for monitoring patients on enzyme replacement therapy (ERT). Data was collected from seventy-eight adults who were referred to the Vanderbilt Program for Metabolic Bone Disease for evaluation of a possible or confirmatory HPP diagnosis between July 2014 through December 2019. Fifty-nine patients were diagnosed with HPP and nineteen were excluded from a diagnosis of HPP. The urine PEA results of those patients with a confirmed diagnosis of HPP and those patients with a diagnosis of HPP excluded were captured and compared to other laboratory and clinical parameters consistent with HPP, including alkaline phosphatase (ALP) activity, plasma pyridoxal 5'-phosphate (PLP), the presence of musculoskeletal abnormalities, and genetic testing for pathogenic mutations in ALPL. Initial urine PEA values in patients in our HPP cohort and not on ERT were significantly higher (median=150.0nmol/mg creatinine, IQR=82.0-202.0) compared patients in our HPP negative group (median 18.0nmol/mg creatinine, IQR=14.0-30.0, p<0.0001) and higher than patients on ERT (median 65.0nmol/mg creatinine, IQR=45.3-79.8). Patients who began ERT had a decline in urine PEA levels after treatment with a mean decrease of 68.1%. Plasma ALP levels were significantly lower in the group of patients with HPP and not on ERT group (median=24.0U/L, IQR=15.0-29.50) compared to the patients without HPP (median=45.50U/L, IQR=34.0-62.0;) and plasma PLP levels were significantly higher in the HPP non-ERT group (median=284.0nmol/L, IQR=141.0-469.4) compared to the patients without HPP (median=97.5nmol/L, IQR=43.7-206.0;). The area under the curve (AUC) of urine PEA, ALP, and PLP to distinguish between HPP and non-HPP patients is 0.968, 0.927 and 0.781, respectively, in our cohort. Urine PEA had 100% specificity (95% CI of 83.2% to 100.0%) for diagnosing HPP at a value >53.50nmol/mg creatinine with a sensitivity of 88.4%; 95%CI 75.5 to 94.9%. ALP had a 100% specificity (95% CI of 82.4% to 100.0%) for diagnosing HPP at a value <30.5U/L with a sensitivity of 77.2%; (95%CI 64.8 to 86.2%). PLP had a 100% specificity (95% CI of 81.6% to 100.0%) for diagnosing HPP at a value >436nmol/L with a sensitivity of 26.9%; (95%CI 16.8 to 40.3%). The most common pathogenic or likely pathogenic mutations in our cohort were c.1250A>G (p.Asn417Ser), c.1133A>T (p.Asp378Val), c.881A>C (p.Asp294Ala), c.1171C>T (p.Arg391Cys), and c.571G>A, (p.Glu191Lys). Urine PEA is a promising diagnostic and confirmatory marker for HPP in patients undergoing investigation for HPP. Urine PEA also has potential use as a marker to monitor ERT compliance. Future studies are necessary to evaluate the association between PEA levels and clinical outcomes.

Dissecting mutational allosteric effects in alkaline phosphatases associated with different Hypophosphatasia phenotypes: An integrative computational investigation.

Hypophosphatasia (HPP) is a rare inherited disorder characterized by defective bone mineralization and is highly variable in its clinical phenotype. The disease occurs due to various loss-of-function mutations in ALPL, the gene encoding tissue-nonspecific alkaline phosphatase (TNSALP). In this work, a data-driven and biophysics-based approach is proposed for the large-scale analysis of ALPL mutations-from nonpathogenic to severe HPPs. By using a pipeline of synergistic approaches including sequence-structure analysis, network modeling, elastic network models and atomistic simulations, we characterized allosteric signatures and effects of the ALPL mutations on protein dynamics and function. Statistical analysis of molecular features computed for the ALPL mutations showed a significant difference between the control, mild and severe HPP phenotypes. Molecular dynamics simulations coupled with protein structure network analysis were employed to analyze the effect of single-residue variation on conformational dynamics of TNSALP dimers, and the developed machine learning model suggested that the topological network parameters could serve as a robust indicator of severe mutations. The results indicated that the severity of disease-associated mutations is often linked with mutation-induced modulation of allosteric communications in the protein. This study suggested that ALPL mutations associated with mild and more severe HPPs can exert markedly distinct effects on the protein stability and long-range network communications. By linking the disease phenotypes with dynamic and allosteric molecular signatures, the proposed integrative computational approach enabled to characterize and quantify the allosteric effects of ALPL mutations and role of allostery in the pathogenesis of HPPs.

Perspective on Dentoalveolar Manifestations Resulting From PHOSPHO1 Loss-of-Function: A Form of Pseudohypophosphatasia?

Mineralization of the skeleton occurs by several physicochemical and biochemical processes and mechanisms that facilitate the deposition of hydroxyapatite (HA) in specific areas of the extracellular matrix (ECM). Two key phosphatases, phosphatase, orphan 1 (PHOSPHO1) and tissue-non-specific alkaline phosphatase (TNAP), play complementary roles in the mineralization process. The actions of PHOSPHO1 on phosphocholine and phosphoethanolamine in matrix vesicles (MVs) produce inorganic phosphate (Pi) for the initiation of HA mineral formation within MVs. TNAP hydrolyzes adenosine triphosphate (ATP) and the mineralization inhibitor, inorganic pyrophosphate (PPi), to generate Pi that is incorporated into MVs. Genetic mutations in the ALPL gene-encoding TNAP lead to hypophosphatasia (HPP), characterized by low circulating TNAP levels (ALP), rickets in children and/or osteomalacia in adults, and a spectrum of dentoalveolar defects, the most prevalent being lack of acellular cementum leading to premature tooth loss. Given that the skeletal manifestations of genetic ablation of the Phospho1 gene in mice resemble many of the manifestations of HPP, we propose that Phospho1 gene mutations may underlie some cases of “pseudo-HPP” where ALP may be normal to subnormal, but ALPL mutation(s) have not been identified. The goal of this perspective article is to compare and contrast the loss-of-function effects of TNAP and PHOSPHO1 on the dentoalveolar complex to predict the likely dental phenotype in humans that may result from PHOSPHO1 mutations. Potential cases of pseudo-HPP associated with PHOSPHO1 mutations may resist diagnosis, and the dental manifestations could be a key criterion for consideration.

Hypophosphatasia.

Hypophosphatasia (HPP) is an inherited metabolic disease caused by loss-of-function mutations in the tissue non-specific alkaline phosphatase (TNAP) gene. Reduced activity of TNAP leads to the accumulation of its substrates, mainly inorganic pyrophosphate and pyridoxal-5′-phosphate, metabolic aberrations that largely explain the musculoskeletal and systemic features of the disease. More than 400 ALPL mutations, mostly missense, are reported to date, transmitted by either autosomal dominant or recessive mode. Severe disease is rare, with incidence ranging from 1:100,000 to 1:300,000 live births, while the estimated prevalence of the less severe adult form is estimated to be between 1:3100 to 1:508, in different countries in Europe. Presentation largely varies, ranging from death in utero to asymptomatic adults. In infants and children, clinical features include skeletal, respiratory and neurologic complications, while recurrent, poorly healing fractures, muscle weakness and arthropathy are common in adults. Persistently low serum alkaline phosphatase is the cardinal biochemical feature of the disease. Management requires a dedicated multidisciplinary team. In mild cases, treatment is usually symptomatic. Severe cases, with life-threating or debilitating complications, can be successfully treated with enzyme replacement therapy with asfotase alfa.

Screening for hypophosphatasia: does biochemistry lead the way?

Patients with childhood hypophosphatasia (HPP) often have unspecific symptoms. It was our aim to identify patients with mild forms of HPP by laboratory data screening for decreased alkaline phosphatase (AP) within a pediatric population. We conducted a retrospective hospital-based data screening for AP activity below the following limits: Girls:≤12 years: <125U/L; >12 years: <50U/L Boys:≤14 years: <125U/L; >14 years: <70U/L. Screening positive patients with otherwise unexplained hypophosphatasemia were invited for further diagnostics: Re-test of AP activity, pyridoxal 5'-phosphate (PLP) in hemolyzed whole blood, phosphoethanolamine (PEA) in serum and urine, and inorganic pyrophosphate in urine. Sequencing of the ALPL gene was performed in patients with clinical and/or laboratory abnormalities suspicious for HPP. We assessed a total of 14,913 samples of 6,731 patients and identified 393 screening-positive patients. The majority of patients were excluded due to known underlying diseases causing AP depression. Of the 30 patients who participated in the study, three had a decrease in AP activity in combination with an increase in PLP and PEA. A heterozygous ALPL mutation was detected in each of them: One patient with a short stature was diagnosed with childhood-HPP and started with enzyme replacement therapy. The remaining two are considered as mutation carriers without osseous manifestation of the disease. A diagnostic algorithm based on decreased AP is able to identify patients with ALPL mutation after exclusion of the differential diagnoses of hypophosphatasemia and with additional evidence of increased AP substrates.

Clinical and Genetic Characterization of Craniosynostosis in Saudi Arabia.

Background: Craniosynostosis (CS) is defined as pre-mature fusion of one or more of the cranial sutures. CS is classified surgically as either simple or complex based on the number of cranial sutures involved. CS can also be classified genetically as isolated CS or syndromic CS if the patient has extracranial deformities. Currently, the link between clinical and genetic patterns of CS in the Saudi population is poorly understood.Methodology: We conducted a retrospective cohort study among 28 CS patients, of which 24 were operated and four were not. Clinical and genetic data were collected between February 2015 and February 2019, from consenting patient's families. The electronic chart data were collected and analyzed including patient demographics, craniofacial features, other anomalies and dysmorphic features, operative data, intra cranial pressure (ICP), parent consanguinity and genetic testing results.Results: The most common deformity in our population was trigonocephaly. The most performed procedure was cranial vault reconstruction with fronto-orbital advancement, followed by posterior vault distraction osteogenesis and suturectomy with barrel staving. Genetics analysis revealed pathogenic mutations in FGFR2 (6 cases), TWIST1 (3 cases), ALPL (2 cases), and TCF12 (2 cases), and FREM1 (2 case).Conclusion: Compared to Western countries, our Saudi cohort displays significant differences in the prevalence of CS features, such as the types of sutures and prevalence of inherited CS. The genomic background allows our phenotype-genotype study to reclassify variants of unknown significance. Worldwide, the sagittal suture is the most commonly affected suture in simple CS, but in the Saudi population, the metopic suture fusion was most commonly seen in our clinic. Further studies are needed to investigate the characteristics of CS in our population in a multicenter setting.

GSK3\u03b2 rephosphorylation rescues ALPL deficiency-induced impairment of odontoblastic differentiation of DPSCs

BackgroundPremature exfoliation of the deciduous teeth is a common manifestation in childhood patients with hypophosphatasia (HPP), which is an autosomal inherited disease caused by ALPL mutations. Dysplasia of the cementum, dentin, and alveolar bone has been proposed to be the main reasons for the exfoliation of teeth, while the extraordinarily complex intracellular mechanisms remain elusive. Dental pulp stem cells (DPSCs) have been demonstrated to successfully regenerate functional pulp-dentin-like tissue. Dental pulp cells derived from HPP patients impaired mineralization; however, insight into the deeper mechanism is still unclear.MethodsThe effects of ALPL on odontoblastic differentiation of DPSCs from HPP patient were assessed by Alizarin Red staining, immunofluorescent staining, Western blot and RT-PCR, and micro-CT assays.ResultHere, we found DPSCs from HPP patient exhibited low ALP activity and impaired odontoblastic differentiation. Meanwhile, we found that loss of function of ALPL reduced phosphorylation of GSK3β in DPSCs. While GSK3β rephosphorylation improved odontoblastic differentiation of HPP DPSCs with LiCl treatment. Finally, we demonstrated systemic LiCl injection ameliorated tooth-associated defects in ALPL+/− mice by enhanced phosphorylation of GSK3β in the teeth.ConclusionsOur study indicates that ALPL regulates odontoblastic differentiation of DPSCs and provides useful information for understanding how ALPL deficiency led to tooth dysplasia and, ultimately, may inform efforts at improvement tooth defects in HPP patients.

Novel mutation in the ALPL gene with a dominant negative effect in a Japanese family.

Hypophosphatasia (HPP) is caused by mutations in the ALPL gene encoding tissue nonspecific alkaline phosphatase (TNSALP) and inherited in either an autosomal recessive or autosomal dominant manner. It is characterized clinically by defective mineralization of bone, dental problems, and low serum ALP levels. In the current report, we demonstrate a novel mutation in the ALPL gene (c.244G > A p.Gly82Arg) in a Japanese family with low serum ALP levels. The ALPL gene analysis using hybridization capture-based next-generation sequencing was performed. The expression plasmids of the wild type and mutated TNSALP were introduced into COS-7 cells. The enzymatic activity of ALP in the cell lysates was measured using p-nitrophenylphosphate as a substrate. TNSALP with the novel ALPL mutation (c.244G > A p.Gly82Arg) completely lost its enzymatic activity and suppressed that of wild-type TNSALP, corroborating its dominant negative effect. The diagnosis of autosomal dominant HPP was confirmed in three members of the family. Our approach would help to avoid the inappropriate use of bone resorption inhibitors for currently mis- or under-diagnosed HPP, given that the presence of further, yet undetected mutations of the ALPL gene are plausible.

Diagnosis of Hypophosphatasia in Adults Presenting With Metatarsal Stress Fracture: Proof-of-Concept for a Case-Finding Strategy.

ABSTRACTHypophosphatasia (HPP) is caused by loss‐of‐function mutations in ALPL resulting in decreased alkaline phosphatase (ALP) activity. Metatarsal stress fracture (MSF) is a common clinical feature of hypophosphatasia in adults. In this study, the primary objectives were to determine whether new cases of ALPL variants could be identified in patients with MSF and who also had serum ALP concentration below the reference range and to phenotype their clinical course. Electronic health records were queried for adult patients with MSF using International Classification of Disease codes (ICD‐9, ICD‐10CM) and ALP measurements. Patients with ALP levels below the normal limit were invited to receive mutational analysis of ALPL and to complete the following surveys: the Short Form 36 version 2 (SF36v2), the Brief Pain Inventory‐Short Form (BPI), and the Health Assessment Questionnaire Disability Index (HAQ‐DI). Cases with and controls without ALPL pathogenic variants were compared by survey scores and clinical variables relevant to fracture. In 1611 patients with MSF presenting to a podiatry clinic (10/1/2011–10/1/2017), 937 had ALP measurement, of whom 13 (1.4%) had ALP levels below the lower normal limit. In eight patients consenting to participate, two had heterozygous pathogenic ALPL variants. ALPL variants were found in 2 of 1611 patients (0.12%) with MSF, 2 patients of 937 (0.21%) in those with MSF and any ALP measurement, and 2 of 13 patients (15%) in MSF and decreased ALP level. Cases versus controls rated lower scores on eight of eight SF36v2 scales (range, 0–100); higher scores for worst pain (8.0 vs. 0.8) and average pain (6.0 vs. 0.7) on the BPI (range, 0–10); and higher standard disability score (1.4 vs. 0) on the HAQ‐DI (range, 0–3). These data provide proof‐of‐concept for HPP case identification in patients presenting to a podiatry clinic with MSF, suggesting a search for historically low ALP levels may be a useful step for consideration of HPP diagnosis, and supports a prospective study to determine an optimal case‐finding strategy. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Clinical and molecular findings in children and young adults with persistent low alkaline phosphatase concentrations.

Hypophosphatasia is a rare inherited metabolic disease resulted by ALPL gene mutations. It is characterized by defective bone and teeth mineralization. The phenotypic spectrum is highly variable ranging from lethal perinatal form to mild forms which are only diagnosed in adulthood or remain undiagnosed despite persistently low concentrations of ALP. The aim of this study is to evaluate the clinical phenotype and frequency of ALPL mutations in a group of patient with hypophosphatasaemia. Thirty individuals with alkaline phosphatase values below 40 IU/L in at least two assessments and having no alternative explanation for their low ALP concentrations were included in the study. The clinical features and radiological data of the study group were re-investigated for hypophosphatasia-related findings. ALPL sequence analysis was performed using Sanger sequencing. No patient in the study group had severe symptoms, nor had they initially been diagnosed as having hypophosphatasia. Four different heterozygous ALPL mutations (c.542C>T, c.648 + 1G>A, c.657G>T and c.862 + 1G>C) were found in four patients. One splice site mutation (c.862 + 1G>C) was reported for the first time in this study. ALPL sequence analysis may help to diagnosing genetic defects in individuals with persistently low ALP concentrations and provide to take preventive measures before symptoms appear. As in the other populations, HPP displays allelic heterogeneity in our population.

Dentoalveolar Defects of Hypophosphatasia are Recapitulated in a Sheep Knock-In Model.

Hypophosphatasia (HPP) is the inherited error-of-metabolism caused by mutations in ALPL, reducing the function of tissue-nonspecific alkaline phosphatase (TNAP/TNALP/TNSALP). HPP is characterized by defective skeletal and dental mineralization and is categorized into several clinical subtypes based on age of onset and severity of manifestations, though premature tooth loss from acellular cementum defects is common across most HPP subtypes. Genotype-phenotype associations and mechanisms underlying musculoskeletal, dental, and other defects remain poorly characterized. Murine models that have provided significant insights into HPP pathophysiology also carry limitations including monophyodont dentition, lack of osteonal remodeling of cortical bone, and differing patterns of skeletal growth. To address this, we generated the first gene-edited large-animal model of HPP in sheep via CRISPR/Cas9-mediated knock-in of a missense mutation (c.1077C>G; p.I359M) associated with skeletal and dental manifestations in humans. We hypothesized that this HPP sheep model would recapitulate the human dentoalveolar manifestations of HPP. Compared to wild-type (WT), compound heterozygous (cHet) sheep with one null allele and the other with the targeted mutant allele exhibited the most severe alveolar bone, acellular cementum, and dentin hypomineralization defects. Sheep homozygous for the mutant allele (Hom) showed alveolar bone and hypomineralization effects and trends in dentin and cementum, whereas sheep heterozygous (Het) for the mutation did not exhibit significant effects. Important insights gained include existence of early alveolar bone defects that may contribute to tooth loss in HPP, observation of severe mantle dentin hypomineralization in an HPP animal model, association of cementum hypoplasia with genotype, and correlation of dentoalveolar defects with alkaline phosphatase (ALP) levels. The sheep model of HPP faithfully recapitulated dentoalveolar defects reported in individuals with HPP, providing a new translational model for studies into etiopathology and novel therapies of this disorder, as well as proof-of-principle that genetically engineered large sheep models can replicate human dentoalveolar disorders. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Dental defects in the primary dentition associated with hypophosphatasia from biallelic ALPL mutations.

ALPL encodes tissue-nonspecific alkaline phosphatase (TNAP), an enzyme expressed in bone, teeth, liver, and kidney. ALPL loss-of-function mutations cause hypophosphatasia (HPP), an inborn error-of-metabolism that produces skeletal and dental mineralization defects. Case reports describe widely varying dental phenotypes, making it unclear how HPP comparatively affects the three unique dental mineralized tissues: enamel, dentin, and cementum. We hypothesized that HPP affected all dental mineralized tissues and aimed to establish quantitative measurements of dental tissues in a subject with HPP. The female proband was diagnosed with HPP during childhood based on reduced alkaline phosphatase activity (ALP), mild rachitic skeletal effects, and premature primary tooth loss. The diagnosis was subsequently confirmed genetically by the presence of compound heterozygous ALPL mutations (exon 5: c.346G>A, p.A116T; exon 10: c.1077C>G, p.I359M). Dental defects in 8 prematurely exfoliated primary teeth were analyzed by high resolution micro-computed tomography (micro-CT) and histology. Similarities to the Alpl-/- mouse model of HPP were identified by additional analyses of murine dentoalveolar tissues. Primary teeth from the proband exhibited substantial remaining root structure compared to healthy control teeth. Enamel and dentin densities were not adversely affected in HPP vs. control teeth. However, analysis of discrete dentin regions revealed an approximate 10% reduction in the density of outer mantle dentin of HPP vs. control teeth. All 4 incisors and the molar lacked acellular cementum by micro-CT and histology, but surprisingly, 2 of 3 prematurely exfoliated canines exhibited apparently normal acellular cementum. Based on dentin findings in the proband's teeth, we examined dentoalveolar tissues in a mouse model of HPP, revealing that the delayed initiation of mineralization in the incisor mantle dentin was associated with a broader lack of circumpulpal dentin mineralization. This study describes a quantitative approach to measure effects of HPP on dental tissues. This approach has uncovered a previously unrecognized novel mantle dentin defect in HPP, as well as a surprising and variable cementum phenotype within the teeth from the same HPP subject.