Clinical presentation of adults with persistently low alkaline phosphatase activity: a retrospective multicentre, cross-sectional study in Germany

Abstract

Objective

Persistently low serum alkaline phosphatase (ALP) activity is the hallmark of hypophosphatasia (HPP). However, low ALP values are not commonly recognised in routine clinical practice, often leading to delayed HPP diagnosis. Determining symptoms associated with persistently low ALP activity may facilitate a timelier diagnosis and improved treatment of patients with HPP. This study aimed to evaluate the signs and symptoms associated with low ALP.

Design

Retrospective, multicentre, cross-sectional study.

Setting

Medical records of adults with low ALP activity collected in 18 German clinics and large medical centres with medical specialty in endocrinology, diabetology, rheumatology and osteology were assessed. Serum ALP activity, medical history, previous diagnoses, laboratory values and symptoms were analysed.

Participants

Records were screened to identify patients≥18 years with ALP activity below the lower limit of the normal range within the last 5 years. Exclusion criteria were oncological or haematological disorders, intensive care at the time of low ALP measurement and having more than one ALP measurement in the normal range or above. Data from 849 patients with ≥1 low ALP value (median age: 44.0, min 18.0, max 90.0), including a subset of 32 patients with documented HPP diagnosis, were analysed.

Results

The study cohort presented with a spectrum of clinical manifestations and diagnostic profiles. Patients with HPP displayed typical symptoms, in particular musculoskeletal pain and fractures, more often than patients without HPP diagnosis (n=817). Among patients without HPP, 26.6% were diagnosed with hypothyroidism. 35 patients displayed 4+ clinical and biochemical signs typical for HPP that were attributed to differential diagnoses, such as rheumatic diseases, fibromyalgia and osteoporosis/osteopenia, suggesting the possibility of underlying HPP in some cases.

Conclusion

Most patients in this study had hypophosphatasemia without further evaluation, highlighting the need for greater awareness of low ALP levels in clinical practice. Recognising low ALP levels, especially when accompanied by symptoms like pain, musculoskeletal and dental abnormalities, is crucial for timely diagnosis and improved patient care.

STRENGTHS AND LIMITATIONS OF THIS STUDY.

• The study data were obtained from a national cohort across multiple centres in Germany, ensuring a balanced representation of patients.

• A retrospective study design over 5 years enabled the collection of a large sample size.

• Alpha Adult is the pioneer study examining the clinical profile of adult patients with low alkaline phosphatase (ALP) levels in Germany.

• The retrospective design prevents establishing causal links between low ALP levels and symptoms, laboratory values, diagnoses or medications, and leads to missing data points that cannot be retrospectively addressed.

• As this is a retrospective study, we were constrained to using data available in existing records, which inherently limits consistency and standardisation across different sites.

Introduction

Alkaline phosphatase (ALP) is a membrane-bound ubiquitous phosphomonoesterase. In humans, four different ALP genes have been identified, coding for the tissue-specific intestinal, placental and germ-cell ALPs, as well as the ubiquitously expressed tissue-non-specific alkaline phosphatase (TNSALP).^{1 2} TNSALP is abundant in skeleton, liver, kidney and developing teeth and is essential for bone and tooth mineralisation.¹,³ Unlike elevated ALP serum levels, which are an established marker for several pathologic conditions, there is little awareness of the implications of low serum ALP activity. Although persistently low ALP activity is the hallmark of hypophosphatasia (HPP)² and crucial for its diagnosis,⁴ following up on low ALP activity is less common in routine clinical practice.

HPP is a rare, inherited, progressive genetic and systemic disorder of bone and mineral metabolism and multiple organ systems. The birth prevalence of perinatal-infantile HPP was described to be 1:300 000 in Europe⁵ and 1:100 000 in Canada⁶ while a considerably higher prevalence was suggested for adults.^{7 8} HPP is caused by loss-of-function mutations in the human alkaline phosphatase gene (ALPL), resulting in low ALP activity.^{2 4 9} HPP is characterised by defective bone and tooth mineralisation, but the clinical presentation can be heterogeneous and may include poorly healing fractures, bone deformities, premature tooth loss, fatigue, musculoskeletal pain and muscle weakness. Neuro-psychiatric, gastrointestinal and kidney problems in adults were described as well.¹⁰,¹³ Seizures and respiratory failure are often associated with severe perinatal and infantile HPP that can have debilitating and life-threatening complications.¹⁴ Adults with HPP have a diminished quality of life and can be strongly impaired in social and occupational areas as well as in daily routines.¹⁵,¹⁷ Many HPP clinical symptoms overlap with those of more common diseases, such as osteoporosis, pain syndrome and rheumatic diseases, potentially resulting in misdiagnosis.⁴¹⁸,²⁰

While there is variability in HPP’s clinical manifestations and age of onset, patients consistently present with reduced serum ALP activity, and usually elevated substrates as serum pyrophosphate (PPi) and pyridoxal-5-phosphate (PLP), the active form of vitamin B6, as well as urinary phosphoethanolamine (PEA).⁹ Given the limited awareness for the clinical significance of low ALP, it is rarely considered, and some laboratories do not flag values below the lower limit of the normal. Interpreting values may be further complicated by missing age-specific and gender-specific reference ranges of ALP, particularly with regard to the lower limits of normal. All these play a role in delaying the diagnosis of HPP.^{10 18 21} Missed or delayed diagnosis also hampers estimating the exact prevalence of HPP.⁷ Poor clinical vigilance or awareness can result in confusion with differential diagnoses, particularly osteoporosis, and in turn lead to the prescription of inappropriate drugs, like antiresorptive agents.²²,²⁴

The current study, ALPHA-Adult (Analysis of the signs and symptoms related to Low alkaline PHosphatase in Adults), was conducted to characterise the clinical presentation of adults with low ALP activity in Germany. The primary objective of this study was to determine the symptoms associated with persistently low ALP in adults, especially in comparison to established HPP symptoms. The secondary objective was to determine the relative incidence of patients with one or more known symptoms of HPP in this population.

Methods

Study design

This study was conducted as a multicentre, retrospective, cross-sectional study to obtain a sufficiently large sample size given that HPP is a rare disease.

Data collection

Data were collected in 18 German clinics and large medical centres with medical specialty in endocrinology, diabetology, rheumatology and osteology between 5 February 2019 and 4 August 2020 (see online supplemental table 1). The number of patients to be included per clinic was restricted to a maximum of 100 patients. Two centres exceeded 100 records by amendment. All patients were included in the analyses.

Data were collected and unidentified by the respective study centres. The study centres entered the unidentified data into the good clinical practice (GCP)-compliant electronic data capture system MARVIN. A randomised centre ID was allocated to each participating site by an independent third party, meaning neither sponsors nor analysts would be able to match data sets to an individual patient or a specific study site.

Laboratory records were screened to identify patients showing reduced total serum ALP activity over a period of 5 years. Normal ranges were based on local standards (see online supplemental table 1). Subsequently, sites checked patient charts for parameters of interest (see section ‘Collected parameters’).

Inclusion and exclusion criteria

To be included, patients had to be ≥18 years with ALP activity below the lower limit of the normal range within the last 5 years. Exclusion criteria were oncological or haematological disorders, intensive care at the time of low ALP measurement and having more than one ALP measurement at the normal or above the normal range. When a single measurement of ALP within or above normal range was classified as an outlier, the respective patient was included. The decision regarding outliers was made by physicians in the respective centres according to good scientific practice. Respective patients exhibited constant low ALP values, with only a single measurement within the normal range.

Collected parameters

Data collection was limited to available patient records. Information on relevant parameters, if available in patient records, was collected by study personnel following best practice standards and entered into the electronic case report form. Patient files were completed by physicians at each site according to best clinical practice.

• Demographic data: among demographic variables collected were age, sex, weight and height at the time of the low ALP serum level detection.

• Medical history: the value of total serum ALP (IU/L), survival status (yes/no) and, if applicable, age at death, selected differential diagnoses of HPP (hypothyroidism, anorexia, zinc deficiency (all recorded as yes/no)), other diagnoses and medication at the time of the low ALP serum level detection.

• Radiological abnormalities: available (yes/no)—deformities, fractures, thorax abnormalities, decreased bone mineral density (BMD) (all recorded as yes/no) and bone mineral density (T-score, Z-score).

• Laboratory values: ALP, PLP, PEA (urine), calcium, phosphate, thyroid-stimulating hormone (TSH), and fT4 (thyroxine) serum levels (all recorded as decreased, normal or increased).

• Organ involvement: kidneys (nephrocalcinosis, hypercalciuria, other kidney disease), lungs (respiratory disorder) and brain/skull (craniosynostosis) (all recorded as yes/no).

• Dental status: premature tooth loss of deciduous teeth, premature tooth loss of permanent teeth, pronounced caries and parodontitis (all recorded as yes/no).

• Pain: muscle, bones, joints and chronic pain in lower extremities (all recorded as yes/no).

• Motor abnormalities: gait, assistive devices (cane, crutches, walker, wheelchair), orthopaedic surgery and difficulties in normal life/daily activities (getting up, household activities, attend) (all recorded as yes/no).

Unavailable recordings were classified as ‘not performed’.

Statistical analyses

All analyses were performed using R software (V.3.6.2, https://www.r-project.org). For descriptive analyses, continuous variables are reported using n (non-missing sample size), median and lower and upper quartiles (Q1, Q3). Categorical variables are reported in frequency and percentages based on the non-missing sample size.

The data are presented for the (1) total cohort (all included patients), (2) HPP group (patients with documented diagnosis of HPP), (3) core group=low ALP unclassified (either no documented diagnosis of HPP (no-diagnosis HPP) or no information on diagnosis of HPP (diagnosis HPP unknown)). The no-diagnosis group and diagnosis-unknown group were combined due to their exhibited similar characteristics and parameters, with no significant statistical differences observed between them.

Further, we investigated the relationship between ALP serum levels and musculoskeletal pain, and motor function in the total cohort descriptively. We only included patients in the analyses for whom information on the respective pain or motor function category and ALP level was available.

Low ALP refers solely to the serum parameter that presents with values below the established reference range. Low ALP can result from various causes, such as hypothyroidism, severe anaemia or treatment with antiresorptive medication. Additionally, persistent low ALP can be attributed to mutations in the ALPL gene, which, when combined with specific clinical signs and symptoms, may explain underlying HPP. To test if low ALP values in the core group (= low ALP unclassified) were explained by conditions other than potentially underlying HPP,²⁵ we examined the incidence of diagnoses (such as hypothyroidism, hypoparathyroidism and coeliac disease) and medication (antiresorptive drugs, steroids/glucocorticoids) that may cause reduced ALP levels.¹³ Main search keywords for examining the incidence of further differential diagnoses are detailed in online supplemental table 2.

We also examined patients in the core group with multiple clinical signs characteristic for HPP more closely to investigate whether there were additional indicators of potentially underlying HPP. Specifically, we examined patients who displayed at least 4 (4+) of the following symptoms: muscle pain, bone pain, joint pain, chronic pain in the lower extremities, history of fractures, skeletal and radiological abnormalities, gait abnormalities and/or walking aid, elevated PLP and/or elevated PEA, and dental abnormalities. Besides general characteristics like age and sex, we focused on differential diagnoses of HPP as misdiagnoses with diseases like fibromyalgia, pain syndromes, osteoporosis or rheumatic diseases are common, as well as various other diagnoses that constitute noticeably frequent comorbidities or help explain the symptomology (eg, hypothyroidism, gonarthrosis or parodontitis).

Analyses were checked for feasibility regarding the number of non-missing levels available or the number of patients in each subgroup. Unfeasible analyses were not performed, in which case only descriptive results are presented.

Patient and public involvement

It was not appropriate or possible to involve patients or the public in the design, or conduct, or reporting, or dissemination plans of our research.

Data were collected from patient records and unidentified by the respective study centres. The study centres entered the unidentified data into the GCP-compliant electronic data capture system MARVIN. A randomised centre ID was allocated to each participating site by an independent third party, meaning neither sponsors nor analysts were able to match data sets to an individual patient or a specific study site. Thus, patients and public were not involved in any way during design recruitment or conduct of the study.

Results

Overall, we collected data from 964 patients; 849 patients passed all inclusion criteria. 115 violated at least one inclusion criterion and were not included in the analysis group (see figure 1). If data were missing for a certain parameter (not filled in the questionnaire), statistical analysis always refers to the number of patients where data for the given parameter was available. For example, HPP status was only available in 488 of 849 cases and 32/488 patients were diagnosed with HPP, resulting in 6.6%.

Figure 1. Patient flow chart depicting data collection: database search, exclusions and inclusions and assignment to the analysis groups. ALP, alkaline phosphatase; HPP, hypophosphatasia.

Characteristics of the total cohort

Out of 849 patients (73.0% female), 32/488 (6.6%) were diagnosed with HPP, while for approximately 50% (n=361) of patients no information on HPP status was available. The median age of the total cohort was 44 years (min 18.0 years, max 90.0 years), the median height was 169 cm (Q1: 164.0, Q3: 174.0), 177.5 cm (Q1: 172.0, Q3: 182.0) for men and 166.0 cm (Q1: 162.0, Q3: 171.0) for women, respectively. The median weight was 68.8 kg (Q1: 59.5, Q3: 80.5) (table 1).

Table 1. Characteristics of the total cohort of patients with low ALP (n=849).

	Value*	Available data points
Demographics and medical history†
Female, % (n)	73.0 (620)	849
Age, median years (Q1, Q3)	44.0 (34.0, 55.0)	849
Height, median cm (Q1, Q3)	169.0 (164.0, 174.0)	761
Weight, median kg (Q1, Q3)	68.8 (59.5, 80.5)	781
ALP in IU/L, median (Q1, Q3)	31.0 (27.0, 37.0)	849
Diagnosis of HPP, % (n)	6.6 (32)	488
Radiological findings‡
Radiological findings available for, % (n)	19.2 (131)	684
Deformities, % (n)	29.3 (29)	99
Fractures, % (n)	22.5 (25)	111
Thorax abnormalities, % (n)	4.3 (4)	93
Decreased BMD, % (n)	17.1 (14)	82
Bone density Z-score, median (Q1, Q3)	−0.7 (−1.5, 0.2)	42
Bone density T-score, median (Q1, Q3)	−2.2 (−2.8, –1.4)	48

^*Results are calculated based on the number of patients with available data points for the respective parameter; patients with missing values are not considered in the respective category. Number of respective available data points for each category is given in the right column.

^†Results are calculated based on the number of patients in the total cohort (n=849).

^‡Results are calculated based on the number of patients with radiological findings (n=131).

ALP, alkaline phosphatase; BMD, bone mineral density; HPP, hypophosphatasia.

Laboratory values (calcium, phosphate, TSH and fT4 values) were within the normal range in over 90% of patients. Median ALP was 31.0 IU/L (Q1: 27.0, Q3: 37.0). Radiological findings were only available for 131/684 patients (19.2%). Overall, bone density was decreased, with an overall T-score of −2.2 (Q1: −2.8, Q3: −1.4), which lies within the range of osteopenia frequently found above an age of 50.^{26 27} Further analysis confirmed that patients with available T-score fall within this age category with a median age of 63.5 years (Q1: 53.5, Q3: 74.5). The Z-score of −0.7 (Q1: −1.5, Q3: 0.2) of the overall cohort was within the normal range. Even though the cohort with a median age of 44 years was relatively young, there was a high prevalence of joint pain (26.8%; 190/708).

Grouped by gender, females (median age: 40.5 years; Q1: 32.0 years, Q3: 49.2 years) were younger compared with males (median age: 55.0 years; Q1: 40.0 years, Q3: 67.0 years) and were diagnosed with HPP more frequently, with 87.5% (28/32) of patients in the diagnosed HPP group being female. In terms of radiological findings (available for 80 female and 51 male patients), females showed a higher percentage of fractures (females: 29.9%; 20/67; males: 11.4%; 5/44) and a higher percentage of decreased BMD (females: 30.2%; 13/43; males: 2.6%; 1/39). Pain symptoms were similar for females and males and ranged between 9.0% (chronic pain in lower extremities; 46/510) and 26.6% (joint pain; 140/527) for females and 11.3% (20/177) and 27.6% (50/181) for males, respectively. We further descriptively compared the correlation between ALP levels and pain and motor function domain, respectively, in the total cohort. Pain status (for any category) and motor/physical function (for any category) were similar, both for patients with ALP level≤40 U/I and >40 U/I (see online supplemental figure 1; respective numbers of patients analysed can be seen in the corresponding figures).

Characteristics of the HPP group versus core group

To better understand the characteristics in the study population, we split the total cohort into two groups: an HPP group (n=32; patients with documented diagnosis of HPP) and a core group (n=817; low ALP unclassified: either ‘no-diagnosis HPP’ or ‘diagnosis HPP unknown’). Given the inhomogeneous sample sizes, no inferential analyses were deemed feasible and hence only descriptive results are reported.

The HPP group comprised 32/849 patients (3.8%). Compared with the core group, ALP levels were lower (median HPP group ALP: 24.5 IU/L (Q1: 19.0, Q3: 29.0); core group median ALP: 31.5 IU/L (Q1: 27.0, Q3: 37.0)) and PLP levels were increased to a higher percentage (HPP group: 100%; 12/12, core group 53.7%; 29/54) (see table 2). Calcium and phosphate levels were in the normal range. The bone density T-score was lower in the HPP group (HPP group −2.9; (Q1: −3.0, Q3: −2.3), core group: −1.9 (Q1: −2.5, Q3: −1.3)). For 13 patients with HPP, information on radiological findings was available, 60% had fractures (6/10). Muscular (47.4%; 9/19), bone (55.0%; 11/20) and joint (73.7%; 14/19) pain were 3–5 times more prevalent compared with the core group (see figure 2).

Table 2. Characteristics of the diagnosed HPP group and the core group (= low ALP unclassified).

	HPP group (n=32)		Core group (n=817)
	Value*	Available data points	Value*	Available data points
Demographics and medical† history
Female, % (n)	87.5 (28)	32	72.5 (592)	817
Age, median years (Q1, Q3)	54.5 (43.8, 68.8)	32	43.0 (34.0, 55.0)	817
Height, median cm (Q1, Q3)	165.0 (159.5, 170.0)	31	169.0 (164.0, 175.0)	730
Weight, median kg (Q1, Q3)	69.1 (57.4, 79.9)	31	68.8 (60.0, 80.5)	750
ALP in IU/L, median (Q1, Q3)	24.5 (19.0, 29.0)	32	31.0 (27.0, 37.0)	817
Radiological findings‡
Radiological findings available for, % (n)	56.5 (13)	23	17.9 (118)	661
Deformities, % (n)	40.0 (4)	20	28.1 (25)	89
Fractures, % (n)	60.0 (6)	20	18.8 (19)	101
Thorax abnormalities, % (n)	0.0 (0)	7	4.7 (4)	86
Decreased BMD % (n)	100.0 (6)	6	10.5 (8)	76
Bone density Z-score, median (Q1, Q3)	−1.1 (−1.3, –0.3)	9	−0.6 (−1.6, 0.2)	33
Bone density T-score, median (Q1, Q3)	−2.9 (−3.0, –2.3)	10	−1.9 (-2.5, –1.3)	38
Laboratory values
Elevated PLP, % (n)	100.0 (12)	12	53.7 (29)	54
Elevated PEA, % (n)	100.0 (1)	1	28.6 (2)	7

^*Results are calculated based on the number of patients with available data points for the respective parameter; patients with missing values are not considered in the respective category. Number of respective available data points for each category is given in the right column.

^†Results are calculated based on the number of patients in the total cohort (n=849).

^‡Results are calculated based on the number of patients with radiological findings (n=131).

ALP, alkaline phosphatase; BMD, bone mineral density; HPP, hypophosphatasia; PEA, phosphoethanolamine; PLP, pyridoxal-5-phosphate.

Figure 2. Prevalence (%) of pain and impairments in motor function and physical activity in the diagnosed HPP group versus core group. HPP, hypophosphatasia.

From the limitedly available records for PLP levels in the core group, PLP measurements were elevated for more than half of the patients (53.7%; 29/54). Other laboratory values were in the normal range. Several patients in the core group displayed typical signs and symptoms of HPP, such as gait abnormalities (35 patients), elevated PLP (29 patients) and premature loss of permanent teeth (7 patients).

Incidence of differential diagnoses associated with low ALP levels

Additionally, we examined the incidence of conditions or medications associated with persistently low ALP levels in the core group (= low ALP unclassified) to assess if low ALP levels could be explained otherwise (table 3). Data in this paragraph were extracted from the questionnaires free text fields ‘documented diagnoses’ and ‘medication’.

Table 3. Differential diagnoses associated with low ALP levels within the core group (n=817).

Differential diagnoses and medication associated with low ALP	Incidence, % (n)
Endocrine metabolic disorders
Hypothyroidism	26.3 (215)
Thyroid hormone treatment (of n=217)	86.5 (186)
Anorexia nervosa	1.1 (7)
Hypoparathyroidism	0.7 (6)
Potential permanent reduction of ALP
Coeliac disease	0.1 (1)
Potential transient reduction of ALP
Steroids/glucocorticoids	12.4 (101)
Antiresorptive drugs (bisphosphonates, denosumab)	1.3 (11)
Deficiency states
Magnesium	0.4 (3)

ALP, alkaline phosphatase.

Of the diagnoses that are published to be associated with low ALP levels,²⁸ hypothyroidism was the most prevalent, comprising a quarter of patients in the core group (26.3%; 215/817). However, further analysis showed that 86.5% (186/215) of these patients received thyroid hormone treatment to compensate for defective thyroid function and normalise ALP levels. This suggests additional factors influencing the low ALP readings in hypothyroidism patients. Steroids/glucocorticoids, which can induce a transient reduction of ALP, were documented for 12.1% of patients (99/817). Antiresorptive drugs and deficiency states (eg, low zinc/magnesium), as well as other conditions associated with low ALP, were rare. Overall, low ALP activity could rarely be associated with a certain condition or medication in most patients. However, data availability was restricted, and patients’ records may not be complete.

Characteristics of patients with 4+ clinical signs typical of HPP

We further examined patients with 4+ clinical signs and symptoms typical of HPP in the core group (= low ALP unclassified) to check for further indicators of a possibly underlying HPP (table 4 and online supplemental table 3).

Table 4. Characteristics of selected patients with 4+ clinical signs typical of HPP (n=35) in the core group.

Patient	Age (years)	Typical characteristics of HPP						Selected diagnoses†
		Muscle pain	Bone pain	Joint pain	History of fractures	Elevated PLP and/or elevated PEA	Dental abnormalities*
1	75	×	×	×	×			Hypothyroidism
3	39	×	×	×		×		Fibromyalgia
4	22	×	×	×		×		Fibromyalgia, arthralgia
5	46	×	×	×		×		Oligoarthritis; hypothyroidism; suspected spondyloarthritis
6	36	×	×	×			×	Fibromyalgia; parodontitis; hypothyroidism; osteitis; s/p jaw surgery
7	74		×	×				Fibromyalgia; osteoporosis; osteoarthritis knees, hands, shoulders
8	63	×	×	×				Hypothyroidism
9	81	×	×	×				Rheumatoid polyarthritis; chronic pseudoradicular lumbar pain syndrome; osteoporosis
10	62		×	×			×	Chronic polyarthritis; hypothyroidism
11	32	×	×	×				Rheumatoid arthritis
14	52	×	×	×				Fibromyalgia
16	73			×				Spondylodiscitis
17	42		×	×				Osteochondrosis and arthrosis; hypothyroidism
18	56	×		×				Spondylophytes
19	36	×	×	×				Somatoform pain disorder
20	62	×	×	×				Fibromyalgia
23	34	×	×	×				Fibromyalgia
24	38	×	×	×				Pain syndrome; hypovitaminosis D; hypothyroidism
25	32	×	×	×				Ankylosing spondylitis
26	52	×	×	×				Chronic polyarthritis
27	63	×	×	×				Onset osteopenia
28	79	×	×	×				Fibromyalgia; hypothyroidism
29	48	×	×	×				Osteopenia
31	58	×	×	×				Osteopenia; spondylosis deformans
33	50	×	×	×				Chronic polyarthritis
34	50	×	×	×				Fibromyalgia; chronic polyarthritis
35	76			×				Coxarthrosis

Full version of this table is provided in the supplemental material (online supplemental table 3).

^*Premature loss of deciduous teeth, premature loss of permanent teeth, pronounced caries and periodontitis.

^†Differential diagnoses of HPP as well as other diseases that elicit similar symptoms or diseases that constitute noticeably frequent comorbidities.

F, female; HPP, hypophosphatasia; M, male; PEA, phosphoethanolamine; PLP, pyridoxal-5-phosphate; s/p, status post.

Overall, 35/817 patients displayed four or more clinical signs typical for HPP (2 patients with six typical symptoms; 11 patients with five typical symptoms; 22 patients with four typical symptoms). The median age was 52 years (min 22.0 years, max 90.0 years) and the majority of patients (77.1%; 27/35) were female. The most common typical symptoms of HPP among the patients were joint pain (97.1%; 34/35), bone pain (88.6%; 31/35), muscle pain (85.7%; 30/35) and chronic pain in the lower extremities (80.0%; 28/35). Further, 15/35 patients (42.9%) reported gait abnormalities and/or the necessity of a walking aid. The most common diagnoses among these patients were inflammatory rheumatic diseases, for example, rheumatoid arthritis and psoriatic arthritis (25.7%; 9/35), fibromyalgia (25.7%; 9/35) and osteoporosis/osteopenia (20.0%; 7/35), which constitute differential diagnoses of HPP, as well as hypothyroidism (25.7%; 9/35), which is a suggested cause of low ALP activity.

Discussion

One potential cause of persistently low serum ALP activity is HPP. The wide spectrum of clinical HPP presentations makes the diagnosis challenging. Diagnostic delay is common due to the rarity of the disease and lack of awareness among physicians, resulting in a median diagnostic delay of 8–12 years in adults.^{17 21 29} This reiterates the value of low ALP activity as a biomarker of the disease in combination with other common HPP clinical symptoms.^{2 4 13}

Despite the autosomal inheritance pattern of the ALPL gene, a significant number of the patients in our cohort were female (73%). This trend is likely attributable to a higher frequency of symptom reporting among women compared with men and is also evident in the cohort data from the global HPP registry.³⁰ The lifetime prevalence of reported fractures ranges from 40% to 55%,³¹ of which 5–10% were reported to be non-healing.^{32 33} In our cohort, with a median age of 44 years, the prevalence of fractures was 22.5%, lying within the expected range regarding the general population lifetime prevalence. Since the number of diagnosed HPP patients in our cohort is rather small (n=32/849), we do not expect them to have a significant influence on the average fracture incidence. The prevalence of joint pain (total cohort: 26.8%) was similar to the general population, in which acute joint pain (last 24 hours) was reported to affect 29.3% of women and 24.4% of men.³⁴

The large difference in group sizes between the HPP group and the core group does not permit inferential analyses, and descriptive results need to be taken with caution. The HPP group showed common HPP symptoms: higher levels of PLP and PEA, poor dental status and reduced motor function compared with the core group. In addition, the HPP group had a multiple times higher prevalence of muscular, bone and joint pain, which is in line with previous data.^{11 15 17 35 36} These findings fit well with literature, suggesting pain to be the most common symptom of HPP.^{15 35} The prevalence of fractures in the HPP group was higher than that in the core group and comparable to other HPP cohorts.^{15 35} In line with literature,² PLP levels were comparatively elevated in the HPP group. These typically result from low ALP activity, leading to substrate accumulation. Interestingly, also a substantial number of patients in the core group (53.7%; 29/54) showed elevated PLP values that could originate from an underlying HPP or be secondary to vitamin B6 treatment, which may not always be documented in the medical records.

Although more prevalent in the HPP group, HPP symptoms were also reported in the core group. These findings are in line with previous studies that described HPP symptoms in patients with low ALP without HPP diagnosis.¹⁸,^{2029 36} Caution is required when comparing the prevalence of HPP symptoms from this study with literature since other studies often included patients with differential diagnoses of HPP, for example, rheumatic patients,²⁰ implicating a higher prevalence of HPP symptomology.

Several conditions are associated with low ALP levels.¹³ Among the examined differential diagnoses of low ALP, hypothyroidism was most prevalent in the core group. However, most patients were receiving thyroid hormone treatment and TSH levels were in the normal range. This should compensate for defective thyroid function and normalise ALP levels, suggesting additional factors that influence the low ALP values in patients with hypothyroidism. The frequent occurrence of hypothyroidism may also be a selection bias related to the high number of endocrinology centres that participated in our study as these may be more likely to identify patients with hypothyroidism. Overall, low ALP levels had no medical explanation in most patients.

Examination of patients in the core group with symptoms similar to HPP frequently revealed differential diagnoses of HPP, such as inflammatory rheumatic diseases, osteoporosis/osteopenia and fibromyalgia. This suggests the possibility of an underlying HPP condition in at least some patients that may explain low ALP levels. Data from the HPP registry demonstrate that diagnosis with inflammatory rheumatic diseases and fibromyalgia is often part of the HPP patient disease history.²¹ This underlines the possibility of HPP being undiagnosed or misdiagnosed for other diseases also in this study’s cohort. It is important to mention that patients can present with an overlay of different underlying diseases, and thus, patients can be correctly diagnosed with fibromyalgia and exhibit HPP at the same time. Along this line, it has been shown in two independent studies that patients can simultaneously present with rheumatic diseases and HPP.³⁷

Our data suggest that low ALP, regardless of its cause, could mimic the symptoms of minimally symptomatic HPP disease, meaning that low ALP itself could be the driver of these symptoms, also in the absence of HPP. However, the absence of a control group characterised by normal ALP levels prevents drawing significant conclusions. Without a comparative baseline established by a control group, it is challenging to attribute the observed symptoms exclusively to deviations in ALP activity. In addition, some of the symptoms reported, such as muscle or joint pain, can be rather common in the general population.³⁸ Nevertheless, a number of patients in the core group presented with one or several HPP symptoms, while only a relatively small proportion of them can be assumed to carry the disease. In this respect, the connection between a low ALP and, for example, musculoskeletal pain must be further examined in the future.

Overall, our findings are well aligned with those reported in literature, both suggesting that an HPP should be considered or ruled out when low ALP levels and corresponding symptoms are present. For example, in a retrospective analysis of medical records in adult rheumatology patients with ALP serum levels below normal ranges, Karakostas et al examined clinical signs and genetic test results for HPP. They describe a subset of patients displaying several clinical symptoms typical of HPP that might have been misdiagnosed.²⁰ Findings from Desborough et al support the use of ALP and PLP measurements along with other demographic and clinical characteristics to be predictive for HPP diagnosis in adults visiting a metabolic bone clinic.³⁹ Along the same line, other studies suggest a higher prevalence of HPP cases than previously published when examining mutations in the ALPL gene among adults with low ALP activity.^{29 35 36 40} Results from Garcia et al suggest that moderate forms of HPP could be twice as prevalent as previously reported in Spanish cohorts.¹⁹ Similar results are noted in children and adolescents,^{18 41} underlining that HPP is an underdiagnosed disease and emphasising the importance of recognising low ALP activity as an important diagnostic parameter in clinical settings.¹⁸

While the physiological role of ALP is not fully understood, accurate diagnosis of HPP is crucial. Bisphosphonates, used for osteoporosis, could impair skeletal mineralisation in HPP and lower ALP levels,^{11 22} making pretreatment ALP measurement essential. When diagnosing osteoporosis, underlying diseases should be ruled out, and reduced ALP should be considered. Persistently low ALP, combined with HPP-like symptoms, should alert clinicians to the possibility of HPP. Despite low ALP not being sufficient for diagnosis, it is an inexpensive test that can guide further investigation. A diagnostic algorithm should include a second ALP measurement, serum-PLP (activated vitamin B6) quantification and, if necessary, ALPL gene testing. Standardised diagnostic criteria for HPP have been published to aid physicians.⁴²,⁴⁴ Early diagnosis and treatment of HPP can limit disease progression, alleviate symptoms and improve quality of life.^{2 10 45}

The main limitation of this study is its retrospective, cross-sectional design, which relies on existing records and can limit consistency and standardisation across different sites. This approach also prevents drawing causal conclusions about the origins of ALP levels in relation to symptoms, laboratory values, diagnoses and medication. Likewise, diagnostic procedures could not be predefined and the data set contained many missing values, including a large proportion of patients with no data on HPP diagnosis, due to missing entries in questionnaires. As such, comparisons between different subgroups may be impeded, and a comprehensive picture of the study population is not possible. Given the retrospective character of the study, follow-up on patients in terms of genetic testing or missing values is not possible. A further limitation is the missing standardisation for lower ALP limits across study sites. Local standards depended on the specific laboratory commissioned by each centre. While the validated local standards varied slightly, they mostly fell within a similar range (40–130 IU/L for men, 35–105 IU/L for women, see online supplemental table 1) and a value below the reference allowed for inclusion in the study.

In summary, this study highlights the importance of following up on low ALP levels in clinical practice. Laboratories need to report and flag increased as well as decreased ALP levels, and physicians should be aware of all conditions associated with abnormal ALP. In addition, typical HPP symptoms were prevalent in the HPP group, underscoring their diagnostic value. Some of these symptoms also occurred in non-HPP patients, many of whom had unexplained low ALP levels. Differential diagnoses of HPP were common among patients with multiple HPP-like symptoms, suggesting the possibility of underlying HPP in some cases. The wide spectrum of HPP manifestations makes diagnosis challenging, but if persistently low ALP levels are detected in combination with elevated PLP or PEA and typical symptoms, such as defective bone and tooth mineralisation and musculoskeletal pain, diagnosis of HPP must be considered.

Data availability statement

Data are available upon reasonable request.