In the present study we investigated the prevalence of ALPL-mutations in a cohort of adult patients with musculoskeletal symptoms, typical for both HPP and rheumatological diseases. Screened patients were treated in the rheumatologic outpatient unit but had inconclusive diagnoses such as fibromyalgia, chronic back pain, pseudogout, calcific tendinitis, osteoarthritis, spondylarthopathy, or undifferentiated connective tissue diseases. Main symptoms were pain in general, back pain, arthralgia, and myalgia. Notably, several patients were treated with disease-modifying anti-rheumatic drugs and did not show any response to therapy. Genetic screening of our patients revealed a high prevalence of heterozygous mutation in the ALPL gene in those with persistently low ALP levels. Every second genetically tested patient showed a mutation in the ALPL gene, indicating a hereditary origin of the musculoskeletal symptoms. Low ALP was detected in 5% of our patient cohort and 14% of these patients presented with musculoskeletal complaints and repetitive low ALP compatible with a HPP diagnosis. Although only mild symptoms were detected in unselected populations with low ALP levels [23, 24] musculoskeletal pain, dental disease, and fractures were associated to ALPL variations . Since musculoskeletal pain was an inclusion criterion in the present study, no significant differences were found between patients with and without ALPL mutation. However, periarticular calcification discriminated both groups. Moreover, fractures, pneumonia, dental disease, and impaired function in childhood were more common in heterozygous mutation positive individuals than in patients with low ALP, but without mutation in the ALPL gene.
Although the absolute number of patients with mutation in the ALPL gene identified in the entire study population is relatively low, it should be noted that HPP is a rare genetic disease with a low prevalence of 1/6370 for mild HPP . In literature the prevalence of ALPL mutations depends on the investigated population and the geographic background [26, 27]. In an unselected population of adults from Spain with unexplained low ALP, ALPL mutations were found in 50% of patients. Musculoskeletal pain was again the predominant symptom in all patients with no difference between patients with and without genetic mutation . In another Spanish cohort of more than 78,000 subjects, persistently low ALP was found in 0.12% of subjects. Fifty-six patients with low ALP and no secondary causes were identified. A mutation in the ALPL gene was found in seven out of 16 tested individuals. Most of them had the diagnosis of osteoarthritis, ankylosing spondylitis, fibromyalgia, or osteoporosis, respectively .
A comparable high prevalence of ALPL mutations were also found by others in more specific cohorts. More than 26,000 adults with endocrinological diagnoses were screened for ALP-levels in Denmark. Fifty-one subjects with persistently low ALP levels were identified after exclusion of risk factors for low ALP. Of these, 24 were genetically tested and more than 50% showed a mutation in the ALPL gene and musculoskeletal pain as leading symptom. PLP levels were higher in HPP than non-HPP subjects .
To date, there is only one study reporting on ALP levels in rheumatologic patients. Retrospective screening of medical records including existing genetical testing for ALPL gene mutation in adult patients with rheumatological diseases and repeatedly low ALP levels was done by a study group from Germany . Persistently low ALP levels were detected in 1.31%, which is higher than reported in non-rheumatologic patients by others . In 13 out of 19 previously genetically tested individuals (68.4%) a mutation in ALPL gene was found. Due to the nature of rheumatologic diseases and HPP, all subjects suffered from musculoskeletal pain. The most common rheumatologic diagnoses in ALPL-positive patients were osteoarthritis, rheumatoid arthritis, spondyloarthritis, and collagenosis. Chondrocalcinosis was not linked to HPP in this study . It remains unclear if the patients in this and our study were misdiagnosed or ALPL gene mutation was a concomitant finding.
In contrast to the above-mentioned studies, other authors found a much lower prevalence of low ALP and ALPL mutations, respectively. In a Brazilian cohort of almost 290,000 chemical tests, only 12 patients with repeatedly low ALP and no secondary reason were identified. None of them showed typical symptoms such as musculoskeletal pain, fragility fractures, or chondrocalcinosis. Genetic testing was not performed. The authors hypothesised a lower HPP prevalence in Brazil compared to other countries .
In a paediatric setting, 393 out of 6,731 screened children had low ALP. The authors state that most of them had a feasible explanation. From the remaining 30 children only 3 had low ALP in combination with elevated PLP and ultimately an ALPL-mutation. One child was diagnosed with HPP, 2 were considered mutation carriers .
HPP is often overlooked in adult patients with typical manifestations. In adulthood, diagnosis is usually made above the age of 40 years. Whereas respiratory and dental symptoms usually occur in childhood, onset of pain and fractures was reported at the median age between 33 and 44 years . In patients with first onset of symptoms in adulthood, symptoms preceded diagnosis by ten years or more [9, 25]. This is in line with our findings. Duration of musculoskeletal symptoms to detection of ALPL gene mutation was more than 14 years in our study. However, 23% of patients had also reported functional impairment in childhood pointing to the fact that HPP can remain undiagnosed for decades.
Although most adults do not qualify for enzyme replacement therapy with asfotase alfa, diagnosis of HPP in adults is still important, because an overlap to osteoporosis may occur. In case of uncertain bone metabolism, anti-resorptive drugs should be avoided. Bisphosphonates and denosumab suppress bone turnover and TNSALP activity and might lead to atypical femoral fractures . More recent data suggest a rather lower risk in HPP patients with heterozygous mutation and lack of histological signs of osteomalacia .
Low ALP is the hallmark of HPP. Nevertheless, reference values for ALP depend on age and sex  and the lower limit of normal is often not stated in lab reports. Moreover, as low ALP is a common finding, secondary reasons must be ruled out. The measurement of natural substrates such as PLP or PEA is an important diagnostic step in patients with persistently low ALP-levels and typical symptoms. In case of repeatedly low ALP levels in combination with increased values for PLP and typical clinical signs such as musculoskeletal pain, HPP must be considered . Due to the lack of accessibility no PLP plasma concentrations were evaluated in our study. Considering that, predictive models and machine learning algorithms identified HPP patients more accurately based on levels of ALP and PLP than ALP alone . Established bone turnover markers are not indicative for HPP  and BMD by means of DXA is not necessarily reduced in HPP, as was shown in our population, and supported by other studies . While not mandatory, genetic testing confirms the diagnosis of HPP because Sanger sequencing and MLPA allow the detection of 95% of all ALPL mutations [15, 36].
It has to be considered, that a mutation in the ALPL gene does not necessarily confirms the diagnosis of HPP. The combination of low ALP, musculoskeletal pain and a mutation in the ALPL gene can be both, mild HPP or a heterozygous carrier state with unspecific symptoms. Moreover, chronic pain, which was used as inclusion criterion in the present study, is a common finding also in the general population. International studies have reported a prevalence of chronic pain between 11% and 51.3% in population-based studies [37,38,39,40]. Especially in the absence of other skeletal and extra-skeletal manifestations such as recurrent fractures, atypical femoral fractures, tooth loss, chondro- or nephro-calcinosis, patients are more likely heterozygous mutation positive individuals, rather than HPP patients. Thus, diagnosis of HPP should be made with caution, in order to not overdiagnoses HPP.
In conclusion, a mutation in the ALPL gene should be considered as a possible explanation 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.