Challenges in Hypophosphatasia: Suspicion, Diagnosis, Genetics, Management, and Follow-Up

Hypophosphatasia (HPP) is a rare inborn error of bone mineral metabolism resulting from loss-of-function variants in ALPL, which encodes the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP) [1]. The condition’s hallmark biochemical signature is low serum alkaline phosphatase (ALP) combined with high pyridoxal-5-phosphate (PLP), a vitamin B6 vitamer [2]. The clinical spectrum of HPP is very wide, ranging from asymptomatic to perinatal lethal [1]. Currently, seven clinical forms of HPP are recognized: benign prenatal, perinatal, infantile, severe childhood, mild childhood, adult, and odonto HPP [1]. Interestingly, non-manifesting forms such as “asymptomatic with biochemical phenotype” have also been described [3‒5].

Perinatal and infantile HPP represents severe forms, characterized by bi-allelic pathogenic ALPL variants. Symptoms manifest in utero or at birth, or within the first 6 months of life, respectively [6]. The deficient TNSALP results in severe bone hypomineralization, causing bony deformities, respiratory distress and failure, hypercalcemia, and vitamin B6-responsive seizures [6, 7]. These HPP forms frequently require admission to intensive care units, predicting an unfavorable prognosis with high mortality in the absence of enzyme replacement therapy (ERT) [8]. The benign prenatal HPP form encompasses detection of deformities in utero or at birth, however, with a favorable prognosis. This form is characterized by a lack of severe postnatal symptoms, along with fetal crowding, normal or improved mineralization on ultrasound, and a normal chest size [9]. The severe childhood HPP presents early in life but after 6 months of age and represents a substantial, yet not life-threatening, disease burden [10]. In these severe forms, diagnosis of HPP is usually straightforward given distinct pathognomonic clinical, imaging, and biochemical features [11].

Later onset forms of HPP, such as mild childhood or adult HPP, typically manifest with nonspecific symptoms like musculoskeletal pain, with adult HPP presenting with metatarsal or pseudofractures, without alterations in serum calcium or phosphate. While most forms of HPP have an “odonto” phenotype (loss of primary dentition before age 4 years), this form also exists as a separate disease entity just affecting the teeth.

The less severe course and substantial variability in phenotype and disease burden makes diagnosis challenging for healthcare professionals, not just pediatricians and adult physicians, also endocrinologists and osteologists with expertise in HPP. This increases the likelihood of misdiagnosis or delayed diagnosis in individuals with less distinctive symptomatology [12, 13]. This review aims to comprehensively explore the clinical challenges surrounding the suspicion of HPP, the diagnosis, genetic confirmation, management, and follow-up of these less distinctly symptomatic forms of HPP.

The initial hurdle in diagnosing HPP is its suspicion, owing to its diverse clinical phenotype. Recognizing the biochemical disease signature in the context of varied manifestations among the broad spectrum of symptoms is essential for healthcare professionals. Table 1 outlines the clinical signs and manifestations in which HPP should be considered.

TNSALP is present in bone, liver, kidney, developing teeth, and other organs [14]. As the most prevalent form of ALP in humans, it constitutes the majority of the total ALP activity measured in serum. TNSALP hydrolyzes pyrophosphate (PPi), a powerful inhibitor of mineralization, into inorganic phosphate (Pi), facilitating the mineralization process [15, 16].

Higher serum ALP levels are recognized in physiological conditions like rapid growth, puberty, or fracture healing (osteogenic activity) and in pathological conditions such as rickets and osteomalacia, bone metastatic diseases, hyperparathyroidism, liver or pancreatic disease [17, 18]. Low ALP levels often escape notice due to limited medical training in differential diagnosis of low ALP [19, 20] and lack of reporting age- and sex-specific ranges by some laboratories, leading to false-normal results [21].

Acute conditions like sepsis, major surgery, or trauma can cause transiently low ALP levels. Persistently low ALP is linked to untreated endocrine disorders (hypoparathyroidism, hypothyroidism, hypercortisolism), nutritional or digestive disorders (hypomagnesemia, malnutrition, celiac disease), hematological issues (anemia, multiple myeloma), or drugs (antiresorptive, clofibrate, vitamin D intoxication). Genetic conditions, including Wilson disease, cleidocranial dysplasia, osteogenesis imperfecta, and HPP among others, also result in persistently low ALP levels. Furthermore, preanalytical errors, such as using EDTA tubes, introduce inaccuracies in ALP measurements [22‒25].

Diagnostic workups for low ALP have been proposed [22, 26]. A key biochemical feature of HPP is increased serum PLP (vitamin B6 vitamer) levels due to the reduced dephosphorylation of PLP to pyridoxine by TNSALP. For accurate measurement of plasma vitamin B6, any pyridoxine supplementation should be discontinued at least 1 week prior to testing. This unique combination of low ALP and high PLP levels serves as the biochemical signature of HPP, essential when considering the diagnosis of HPP.

HPP is marked by the accumulation of PPi due to insufficient TNSALP activity in osteoblasts. Excess PPi leads to hypomineralization (rickets and osteomalacia) and other bone manifestations. The varied spectrum of bone symptoms and signs during the life span encountered in HPP poses challenges in identification outside the pediatric disease spectrum.

In the pediatric age-group, rickets and osteomalacia are present in severe perinatal or infantile HPP [1, 27]. This encompasses bone deformities, beading of costochondral junctions, genu valgum/varum, or short stature [28]. Additionally, premature closure of cranial sutures may result in craniosynostosis [29]. Childhood and juvenile HPP may present with chronic musculoskeletal pain and less frequently recurrent fractures with poor healing but not bone deformities or short stature [28]. In adult-onset HPP, individuals commonly manifest symptoms and signs of osteomalacia and bone fragility, such as recurrent metatarsal fractures, femoral pseudofractures (looser zone fractures), or atypical femoral fractures [30, 31]. Poorly healing fractures and remnants of childhood disease such as short stature, genu valgum/varum, or tooth loss may also be observed [32].

Clinicians should conduct thorough diagnostic assessments, including X-rays of wrist/knees in children, and measurement of serum mineralization markers, including ALP, in any subject presenting with skeletal symptoms. Suspected cases of rickets with ALP levels below the reference range should prompt consideration of HPP.

Excluding the severe perinatal and infantile forms, individuals with HPP commonly exhibit a greater number of extraskeletal symptoms compared to skeletal symptoms [27, 33]. A distinctive and nearly universal feature of all pediatric-onset forms of HPP is the early loss of some primary teeth, typically with intact roots, occurring before the age of 4 [28]. Other common extraskeletal symptoms involve neuromuscular features, including muscle weakness and pain, gross motor and cognitive delays, and abnormal gait. Fatigue, headaches, or sleep disturbances have also been reported [34]. Nephrocalcinosis can be present at diagnosis, with or without hypercalcemia, hypercalciuria, and hyperphosphatemia. In adult HPP, ectopic calcification including calcium pyrophosphate dihydrate deposits contribute to pyrophosphate arthropathy (pseudogout), potentially leading to initial consultations with rheumatology units [35, 36].

Historically, HPP diagnosis required confirming persistent hypophosphatasemia (low serum ALP) and conducting a comprehensive assessment, including clinical history, physical examination, laboratory studies (elevated serum PLP), and radiological imaging (rickets, metaphyseal lucencies, pseudofractures in the proximal medial subtrochanteric region of the femur, metatarsal stress fractures) [2, 23]. However, there was no standardized assessment of the relative significance of these various aspects in HPP diagnosis.

Very recently, two groups have proposed clinical criteria for diagnosing HPP. A HPP International Working Group has introduced major and minor criteria for HPP diagnosis through literature reviews and expert opinions (Table 2a) [32, 37]. These criteria deem persistently low ALP as indispensable, prioritizing laboratory and radiological examinations over clinical findings. The group opted for making pathogenic or likely pathogenic variants of the ALPL gene a major criterion, but not obligatory, which takes into account the unavailability of genetic testing in certain parts of the world, or cryptic variants escaping next-generation sequencing techniques.

The ALPL Gene Variant Consortium published a phenotypic score to assess the likelihood of HPP in individuals with identified variants in ALPL (Table 2b) [4]. This score, based on unambiguous clinical signs of the disease, is utilized to contribute to the classification of ALPL variants using the strict criteria of the American College of Medical Genetics and Genomics [38]. The minimum criterion is the presence of the biochemical HPP signature, with a score above 2 indicating a higher likelihood of HPP.

While these proposals for a formal diagnostic framework constitute a significant step, these criteria require validation and evaluation for further refinement. Alternative examinations such as dual X-ray absorptiometry or lateral spine radiography have not demonstrated significant correlation or diagnostic improvement for HPP diagnosis since low bone density or vertebral fracture are not typical features of HPP [39, 40]. Consequently, they are not deemed essential for diagnosis or monitoring, and their utilization should be individualized based on specific clinical circumstances (i.e., postmenopausal status).

HPP is a heritable disorder caused by pathogenic ALPL variants, showcasing extensive allelic heterogeneity with over 450 related variants currently identified [4]. Its inheritance can be autosomal recessive or dominant. In heterozygous cases, mutated monomers may impair wild-type counterparts, reducing ALP activity via dominant negative effects [1]. Severe HPP forms often result from homozygosity/compound heterozygosity, moderate forms from missense variant dominant negative effects, and milder forms from haploinsufficiency mechanisms [41]. However, significant phenotypic variation persists even among patients with identical compound heterozygous genotypes, a complexity yet unexplained [42]. Figure 1 illustrates this variability in two siblings with the same ALPL variants.

The Global ALPL Gene Variant Consortium tackles the challenges posed by variants of unknown significance (VsUS) in ALPL. This open-access database, accessible at https://alplmutationdatabase.jku.at/, currently houses data on 446 ALPL variants and 797 genotypes, serving as a valuable resource for interpreting the clinical implications of reported ALPL variants.

The database features comprehensive tables of nucleotide variants encompassing all reported ALPL variants. It also offers a submission platform for clinicians and geneticists to submit VUS in ALPL for evaluation and reclassification.

An international consortium comprising experts from diverse backgrounds collaborates to form a standardized approach for reclassifying VUS. This multistep variant classification process adheres strictly to the stringent ACMG/AMP variant classification guidelines. The process includes a detailed clinical assessment of the patients’ phenotype, with utilization of the HPP likelihood score. Subsequent steps involve in-depth literature reviews facilitated by AI technology, genetic assessment utilizing extensive resources and predictive tools, and, where needed, in vitro functional testing of residual ALP activity conducted at the Johannes Kepler University Linz (Austria) research laboratory [4].

The Global ALPL Gene Variant Project and its associated database are designed to serve the global medical community, providing a reliable repository of information for clinicians and geneticists managing HPP patients who can look up all genotypes and associated phenotypes for every ALPL variant. The database offers detailed insights into ALPL variants, including evidence supporting their pathogenicity, thereby enhancing diagnostic precision and prognostic accuracy in HPP cases.

The clinical presentation of HPP can vary widely despite identical ALPL genotypes, complicating accurate prognosis and treatment decisions. Thus, comprehensive clinical assessments of individual phenotypes are crucial for guiding management strategies, emphasizing the importance of phenotype-driven approaches over exclusive reliance on genetic data. Nonetheless, genetic testing remains integral for diagnosing suspected HPP cases due to its genetic origin. The absence of detectable ALPL variants should alert clinicians to consider alternative diagnoses, even though cryptic ALPL variants may escape detection by whole-exome sequencing.

Asfotase alfa (Strensiq^®; Alexion Pharmaceuticals, Inc., Boston, MA, USA) is a recombinant, mineral-targeted, human TNSALP administered subcutaneously, which was approved in 2015 as the first treatment for HPP [23]. Explored across a broad age spectrum, its efficacy is well established in severe forms like perinatal and infantile HPP, with improvements in clinical and radiological outcomes and survival [8]. Positive impacts on growth, motor function, and quality of life have also been observed [43‒47]. However, determining the indication for ERT in milder HPP cases presents a challenge, in addition to cost-benefit considerations.

Ensuring a definite diagnosis is fundamental before considering ERT. Other causes of low serum ALP and other musculoskeletal disorders with similar presentations must be excluded (see above). The identification of pathogenic ALPL variants with or without a biochemical HPP signature is insufficient to consider ERT in the absence of significant clinical symptoms.

Treatment indication and initiation, in particular for milder cases, should be conducted in tertiary centers with expertise in HPP, where factors such as genetics, biochemical phenotype, and symptom severity can be interpreted (Table 3) [27, 48‒50]. Decision to commence treatment should be jointly made by the clinical team and the patient, with thorough consideration of potential side effects (e.g., ectopic calcifications, lipoatrophy) [51]. Re-evaluation of treatment efficacy and safety, e.g., at 12 months, is required.

Given the complex nature and wide spectrum of HPP symptoms, a multidisciplinary approach is essential. The core team should comprise endocrinologists, orthopedic surgeons, dentists, complemented by specialized nurses, physiotherapy, occupational therapy, and rehabilitation experts. The involvement of other professionals may vary depending on the patient’s age and specific needs. In perinatal and infantile HPP, collaboration with intensive care specialist, pulmonologists, pediatric neurologists, and neurosurgeons is required for optimal outcomes [11, 23, 52, 53]. In adulthood, relevant specialists may be rheumatologists and immunologists [54, 55].

Monitoring HPP patients under ERT involves three main components to ensure treatment efficacy and safety: laboratory analysis, physical examination, and radiological imaging [50, 55]. Laboratory parameters include disease markers (ALP, plasma PLP, urine phosphoethanolamine), parathyroid hormone (PTH), 25-hydroxy vitamin D, routine blood tests (blood count, renal and liver function, electrolytes). Analyses should be performed at baseline, 3 months, 12 months, and then annually [55]. Clinicians should be aware of potential hypocalcemia with elevated PTH (hungry bones), especially at the start of ERT, and ensure sufficient vitamin D levels and calcium intake [47, 56]. For accurate interpretation of PLP during ERT, adding ALP inhibitors (levamisole) to samples is recommended to prevent in vitro degradation in the presence of ERT. Results must be interpreted considering this possible source of preanalytical error [55].

In children and adolescents with open growth plates, annual radiographic monitoring of the wrists is advised. Knee X-rays are recommended annually if signs of rickets are present, or biannually in the absence of pathology. This evaluation includes assessment of rickets using the Rickets Severity Scale (RSS; 0 = absence of rickets, 10 = severe rickets) and the Radiographic Global Impression of Change scale (RGI-C; −3 = severe worsening, +3 = complete or near-complete healing), as well as bone age assessment [55‒57]. Additional comprehensive skeletal studies may be indicated, as required.

Clinical assessments of pain and motor function are important and require time commitment from the therapy team and patients [55]. Regular evaluation includes mobility (6-min walk test, gross motor function measure), muscle strength (dynamometer, grip and pinch strength), or pain (faces pain scale). Younger children require monitoring of growth and motor milestones (Bayley Scales of Infant and Toddler Development III) every 3 to 6 months. Other aspects like respiratory function or dental health should be assessed at baseline with variable follow-up based on clinical judgment. Dual X-ray absorptiometry follow-up in HPP patients is controversial [39, 40].

The primary response criteria involve skeletal improvement within 3–6 months of initiating ERT, improvement in bone mineralization, or the presence/recurrence of clinical symptoms in both pediatric and adult patients [47, 55]. Other parameters, such as growth rates and head growth, have also been proposed for pediatric cases [50]. For less symptomatic forms of HPP, improvements in pain, locomotion (e.g., 6-min walk test) and biochemical markers (e.g., rise in PTH if suppressed, drop in PLP if elevated) should be used to assess treatment efficacy.

Dose adjustments should account for signs of inadequate response, under- or overtreatment. Undertreatment would reflect biochemically through elevated PLP, hypercalcemia, increased urine Ca/Cr, suppressed PTH, nephrocalcinosis, and, in children, poor radiological response (e.g., RGI-C < +2) [46, 47]. Of note, patients with severe forms may exhibit delayed or partial responses due to preexisting conditions [47]. Overtreatment, harder to detect due to delayed ectopic calcification, justifies concerns, especially considering bone physiology changes post-growth cessation.

In cases of inadequate response, treatment necessity should be reassessed, and potential secondary factors contributing to treatment failure, such as nutritional deficiencies or scoliosis, should be evaluated [55]. A standard asfotase alfa dose of 2 mg/kg thrice weekly, effective in puberty, may constitute a potential overdose post-peak bone mass, necessitating personalized dosing. Discontinuation should be considered in less symptomatic HPP cases with poor response, followed by continued monitoring post-therapy cessation.

Discontinuation of treatment has been reported in the literature, often due to low adherence, leading to clinical worsening (rickets, pain, reduced mobility) [58, 59]. Clinical worsening in milder cases will depend on the original level of severity, remaining growth potential, and length of observation as treated bone is being replaced by osteomalacic HPP bone through remodeling.

Serum ALP values are typically elevated in patients on ERT (>3,000 U/L) [46]. Lack of elevation may indicate nonadherence. Although neutralizing anti-drug antibodies have been detected during ERT, there is limited evidence to comprehend the impact of immune response against asfotase alfa. Also, their measurement is unavailable commercially and access is only through the HPP Registry (http://hppregistry.com).

Currently, there is a notable absence of scientific literature on this milder spectrum of disease. This group may encompass subjects with low ALP without an identifiable cause, or those with a biochemical HPP signature with a pathogenic ALPL variant or a VUS but no clinical symptoms. These individuals may remain asymptomatic or develop symptoms later in life. In the absence of guidelines, we recommend annual monitoring of growth, muscle function (strength, milestones, gait), and pain in children and adolescents, aligning with the protocol for patients undergoing ERT. For adults, monitoring pain and muscle function every 1 to 2 years is advisable.

Bisphosphonates, an established treatment for osteoporosis, are synthetic analogs of PPi [60]. These compounds resist hydrolysis by ALP, theoretically leading to increased PPi levels and potentially exacerbating symptoms of HPP. Despite some reported atypical femur fractures, there is ongoing controversy regarding the recommendation against their use in patients with HPP [61, 62]. This is particularly relevant in postmenopausal women carrying an ALPL variant who develop osteoporotic vertebral fractures typical for estrogen deficiency but not characteristic of HPP. A balanced view and focus on pathophysiology is required for treatment decisions in these cases.

Vitamin D sufficiency should be aimed for in HPP. However, excessive vitamin D supplementation is not recommended [23] as it has the potential to exacerbate hypercalcemia and hypercalciuria, especially in more severe forms [63, 64]. HPP patients with neonatal seizures respond to pyridoxine. Interestingly, upon transitioning to ERT, many patients find they no longer need ongoing pyridoxine or other antiepileptic medications.

Managing HPP poses significant challenges for clinicians. Familiarity with potential disease indicators, the latest diagnostic criteria, genetic aspects, treatment options, and follow-up requirements is crucial for effective patient care. Complexities in handling milder cases, defining treatment response, and avoiding over- or undertreatment demand clinical expertise. A deeper understanding of HPP’s pathophysiology and clinical manifestations, alongside continuous development in integrating diagnosis and treatment approaches, is crucial for improved patient outcomes.

R.M.-L. and F.H. have no conflicts of interest to declare. M.R.F. is funded through a research grant from Alexion and AstraZeneca Rare Disease and paid by Johannes Kepler University, Linz, Austria. V.S. is the UK Chief Investigator for the Global HPP Registry and has received honoraria from Alexion and AstraZeneca Rare Disease. W.H. is a consultant for and has received research funding and honoraria from Alexion and AstraZeneca Rare Disease.

No funding received for this article.

R.M.-L.: conceptualization, investigation, writing – original draft, and writing – review and editing; M.R.F. and F.H.: writing – original draft and writing – review and editing; V.S.: data curation and writing – review and editing; and W.H.: conceptualization, writing – review and editing, and supervision.