Mild hypophosphatasia may be twice as prevalent as previously estimated: an effective clinical algorithm to detect undiagnosed cases

Introduction

Hypophosphatasia (HPP) is a rare genetic disease caused by a loss-of-function mutation in the gene encoding tissue-nonspecific alkaline phosphatase (TNSALP) [1], [2], [3]. TNSALP is involved in the dephosphorylation of a wide range of substrates, including inorganic pyrophosphate (PPi), phosphatidylethanolamine (PEA), pyridoxal-5′-phosphate (PLP) [4], [5], [6], [7], and others such as adenosine triphosphate (ATP), di-phosphoryl lipopolysaccharide (LPS), and phosphorylated osteopontin (p-OPN) [8], [9], [10], [11]. HPP is considered an innate error of metabolism with highly variable clinical presentation, and HPP is usually involved in bone and tooth mineralization abnormalities [11, 12].

Previous studies have shown that the severity of the disease is inversely related to the age at which symptoms appear (except for benign HPP). Thus, perinatal lethal and infantile HPP are the most severe forms, while affected adults have milder HPP forms, with premature tooth loss (odontohypophosphatasia) [13] and increased risk of fractures, chronic pain and joint inflammation, including, in some cases, neurological disorders [14], [15], [16], [17], [18], [19]. Although the main HPP-related symptomology is bone deterioration, this disorder shows great variability in its clinical presentations due to its great genetic heterogeneity (more than 500 mutations have been described in the ALPL gene to date) [20, 21]. In this context, there is increasing evidence of patients with symptoms other than the typical pathognomonic signs of HPP, so this disease is being increasingly considered a multisystemic disease [9, 17, 22, 23].

In terms of prevalence, HPP is currently classified as an ultrarare disease since several studies describe low rates of affected individuals around the world [2, 14, 24]. However, studies previously conducted by García-Fontana et al. and Mornet et al. [12, 22] have shown that HPP may have a higher prevalence than originally estimated, being underdiagnosed in clinical practice due to the overlap of its clinical symptoms with those of other more prevalent diseases, such as osteoporosis or osteomalacia [25, 26]. This fact has important consequences since the use of some antiosteoporotic drugs in HPP patients, such as bisphosphonates that remain for a long time in the body, worsens the prognosis of the disease [18, 27], [28], [29].

Currently, low ALP levels are not considered to be a relevant symptom of any major disorder, so HPP often remains unrecognized. In this context, the clinical laboratory plays a crucial role in the diagnosis of HPP to avoid the underdiagnosis of this disorder. Thus, the reporting of decreased values below normal limits for ALP is a recent update to clinical practice at University Hospital Clínico San Cecilio of Granada.

The recent availability of an effective HPP treatment based on the enzymatic replacement of ALP has generated the need to establish better diagnostic algorithms for early detection of the infant-juvenile form, as well as to re-evaluate those patients with high clinical suspicion of HPP who may have been misdiagnosed with other diseases.

The proactive search for this disorder by means of a structured and systematized analysis will make it possible to know the real prevalence of HPP in each population and to establish appropriate preventive and therapeutic measures.

In this context, the aim of this study was to design and assess the usefulness of an effective protocol to improve the diagnosis of HPP that can be implemented in clinical practice for the early and reliable detection of HPP. In addition, we intended to further investigate the extraskeletal effects of low ALP levels in the affected population to better characterize this disease.

Materials and methods

Diagnostic algorithm to identify potential HPP cases

A diagnostic algorithm was designed and implemented in the Clinical Analysis Unit of the University Hospital Clínico San Cecilio of Granada for the early detection of HPP.

The HPP diagnostic protocol starts through the laboratory computer system database, identifying ALP assessments below the reference values based on the reference ranges shown in Table 1.

When there are two or more ALP results in the medical record and one of them remains near the reference values, the ALP levels are analyzed in future analyses.

If there is only one determination of serum ALP levels in the medical record, and the level is below the reference values, a new determination is requested to check whether the decreased ALP levels are persistently maintained.

In the case of persistently low ALP values, secondary causes of hypophosphatemia [32], [33], [34], [35], [36] are ruled out. After this, the patients with suspected HPP are referred to the pediatric or endocrinology unit, where the patients are informed about HPP disease and the procedure to be followed. Two blood samples are collected from participants to determine their serum PLP levels and to perform Sanger sequencing of the ALPL gene if PLP levels are above the reference values (3.6–18 ng/mL) to identify possible genetic variants associated with HPP (Figure 1).

Figure 1:

Diagnostic algorithm to identify potential HPP cases.

Results

Results of the application of the diagnostic algorithm

Initially, 64,013 patients with ALP determinations in the 30-month period from 1 June 2018 to 31 December 2020 were selected (62,412 subjects in the adult population and 1,601 subjects in the pediatric population). Of these, a total of 765 subjects with decreased ALP levels were selected following the diagnostic algorithm described previously. Among them, 522 subjects were excluded because they had only a single ALP determination. Of the resulting 243 subjects (204 in the adult population and 39 in the pediatric population), 128 were excluded for having two or more ALP values in or above the reference range. After studying the medical records of the remaining 115 patients, 54 subjects were excluded for having possible causes of secondary HPP. Finally, only 24 of the 61 subjects with suspected HPP participated in the study (21 adult subjects and three pediatric subjects) (Figure 2).

Figure 2:

Flowchart of the recruitment of patients potentially affected by HPP. ^aExcluded for having only a single ALP determination. ^bExcluded for having two or more ALP values in or above the reference range. ^cExcluded for having possible causes of secondary hypophosphatasia. ^dExcluded for refusing to participate in the study.

The ALP and PLP levels of the 24 patients who agreed to participate in the study were determined. Analyzing the values observed in the adult population (n=21), we observed means of 24.8 (±7.1) U/L and 91.6 (±106.0) ng/mL, for ALP and PLP respectively. Considering all participating subjects (n=24), 17 adults and three children had persistently decreased ALP activity and increased PLP levels compared to baseline values. These 20 patients underwent genetic screening.

Discussion

Since HPP is a little-known disease and low serum ALP levels are not usually considered in clinical practice as indicators of major disorders, HPP is often underdiagnosed, as we have suggested in previous studies [22]. In this study, a new HPP detection algorithm was proposed by determining low ALP levels using the values stratified by age and sex as established by the standards of the CALIPER study for the pediatric population and the IFCC study for the adult population.

After clinical characterization and genetic testing of the patients, 16 of the 24 study participants (14 adults and two pediatric individuals) showed a mutation in the ALPL gene. According to these data, more than half of the studied population (66.67 %) was not correctly diagnosed following standard clinical practice. If we extrapolate these data to the current population of Spain, this means that 10,779 potential cases of HPP exist that are not currently diagnosed. Considering the proportion of potential HPP patients who did not participate in this study for different reasons (60 %) and the proportion of patients with positive genetic results in the present study, the current estimate of potential undiagnosed HPP cases in Spain could be as high as 27,177 cases, which means that the current HPP prevalence could be 1/1,692 for mild forms. In this context, in 2011, Mornet et al. estimated a mild HPP prevalence of 1/6,370 for Europe [14]. In our previous study in 2019, we estimated a mild HPP prevalence for Spain that duplicated the European mild HPP prevalence estimated by Mornet (1/3,100) [22], which was similar to the new estimation calculated by Mornet et al. in 2021 for Europe (1/2,430) [12]. Thus, in this study, using our proposed diagnostic algorithm, the new estimated prevalence of mild HPP was almost double that previously estimated by our group (1/1,692 vs. 1/3,100).

Regarding ALP and PLP levels related to clinical manifestations in the adult population, we did not observe a link between the severity of clinical manifestations and lower levels of ALP. However, PLP levels could act as a marker of HPP severity, since patients with levels close to 200 ng/μL or higher showed more severe symptomatology (Patients 19, 21, Table 2).

The findings of our study indicate the great importance of establishing a good diagnostic algorithm for the diagnosis of HPP, considering appropriate reference values standardized by age and sex in the laboratory, together with the need to detect and report patients with persistently decreased ALP values in different analytical tests. Indeed, we propose a new algorithm capable of detecting most of the undiagnosed cases, which could improve the quality of life of up to 27,177 people only in Spain. Most importantly, our algorithm could avoid the negative effects of inappropriate treatments such as bisphosphonates, which lead to a worsening of HPP symptomatology. It is important to emphasize the need to perform genetic studies in patients with biochemical analytical values compatible with HPP even in the absence of clinical symptoms, since these patients could have asymptomatic HPP at the current stage, to avoid the use of these mentioned counterproductive therapies that worsen the patients’ prognoses.

Moreover, it should be noted that despite no mutation in the ALPL gene being detected in four patients (patients 2, 3 and 9 (Table 2) and 1 (Table 3)), we cannot rule out an HPP diagnosis since their biochemical levels of ALP and PLP and main clinical manifestations were consistent with a potential HPP diagnosis. The cases of these patients could be explained by the presence of unidentified mutations in the noncoding region of the ALPL gene, since in clinical practice, genetic testing through Sanger sequencing sequences only the coding and flanking regions of the ALPL gene, so the promoter regions or intronic regions of the gene are currently not considered [37]. In this context, mutations in the noncoding regions can also lead to decreased protein activity due to alterations in alternative splicing processes, as we have observed in some patients in whom mutations in the intronic flanking regions have been identified (patients 13 and 14 (Table 2)). On the other hand, the existence of genetic variants in other factors involved in TNSALP regulation, such as RUNX2 and ENPP1 [31, 32], could affect ALP levels in these patients, leading to HPP development through alternative pathways. In this line, there are some diseases, such as acrodermatitis enteropathica, that lead to hypophosphatemia due to Zn²⁺ deficiency caused by a mutation in the SLC39A4 gene encoding the zinc transporter Zip4 [38]. Adequate Zn²⁺ levels are essential for TNSALP activity, so diseases other than HPP can directly affect TNSALP activity, leading to clinical manifestations related to HPP. In this context, we identified two subjects with hemochromatosis in our cohort. Hemochromatosis is a disease recently identified as a secondary cause of HPP due to the iron and ferritin inhibition of ALP activity [34, 36], but it is not currently included in the exclusion criteria considered in clinical practice, which should be updated. In this regard, on the basis of these findings, we have updated our HPP diagnostic protocol considering these secondary causes (hemochromatosis and acrodermatitis enteropathica) as exclusion criteria for HPP diagnosis.

Based on these considerations, HPP prevalence could be even higher than what we are detecting with the applied algorithm. In fact, our group is currently assessing these factors to deepen the understanding of the molecular pathways involved in HPP. In this context, because mutations in ALPL noncoding regions or in other TNSALP regulatory genes have been found, it would be advisable to improve clinical diagnostic protocols by including all known secondary causes of HPP and to perform complete sequencing of the ALPL gene through next-generation sequencing (NGS) to identify mutations in the noncoding regions or in the regulatory sequence of the gene in clinical practice [10, 39].

On the other hand, one of the most important findings of this study is the remarkable incidence of autoimmune diseases observed in patients with low decreased ALP activity. In this context, we found that 9/24 (37.5 %) participants (with persistent low levels of ALP based on our diagnostic algorithm) were affected by some type of autoimmune condition. For example, we found subjects affected by rheumatoid arthritis (patient 5, Table 2); dermatomyositis (patient 9, Table 2); psoriatic spondyloarthrosis and thrombocytopenic purpura (patient 10, Table 2); psoriasis (patient 12, Table 2); lupus (patient 13, Table 2); polyarthritis and synovitis, acne, pustulosis, hyperostosis, osteitis (SAPHO) syndrome (patient 14, Table 2), autoimmune thyroiditis (patient 15, Table 2); juvenile idiopathic arthritis (patient 19, Table 2); and Crohn’s disease (patient 3, Table 3). Among them, considering only the subjects with some mutation in the ALPL gene identified by sequencing in our study, we observed the same incidence (37.5 %), since 6/16 subjects were affected with one of these autoimmune disorders (patients 10, 13, 14, 15, 19 – Table 2, and patient 3, Table 3). Since the prevalence of autoimmune disease in the general population worldwide is estimated to be between 3 and 8 % [40], the high proportion of HPP patients showing these comorbidities (four times higher than the general population) suggests that there might be a relationship between HPP and these conditions. This suggested link between low ALP levels and autoimmune involvement is an important finding not described in the scientific literature to date and has important clinical and therapeutic implications. According to these findings, our research group is currently investigating this issue to better characterize HPP disease and improve the management of the disease at the clinical level.

Regarding the limitations of the study, the main limitation is that it was carried out at a provincial level. Our prevalence calculations are based on an estimation through the extrapolation of national data, considering that the prevalence identified in Granada is similar to the prevalence in the other provinces of Spain. Therefore, to verify this estimated prevalence, a multicentric study of HPP in collaboration with other centers and clinical units from other cities is necessary. It would be interesting even at the European level to collaborate with different hospitals from Europe to establish a real European prevalence estimation. Another limitation of our study is that our algorithm excludes those patients with PLP levels in the range of the reference interval. This is a limitation because some cases of HPP-affected patients with PLP levels in the reference range have been recently found (results to be published). This implies that our algorithm should be updated considering finding. On the other hand, the main strength of our study is the design of an algorithm that has made it possible to detect a high percentage of cases that are usually undiagnosed through routine clinical practice (up to 80 %) in a simple and affordable way for any hospital or health care center. Thus, we can say that our proposed diagnostic algorithm shows high efficacy in detecting HPP and could be easily implemented in clinical practice.

In summary, the findings of this study have a great impact on clinical practice since the application of a diagnostic algorithm as effective as the one presented here can contribute to the correct diagnosis of a high proportion of patients with HPP who are currently undiagnosed or misdiagnosed. This is important since despite being a rare disease, when recalculating the HPP prevalence based on our results, the prevalence is twice as high as the last European estimated prevalence. At the therapeutic level, this knowledge may avoid the use of inappropriate treatments that may worsen the diagnosis of patients. On the other hand, it is worthwhile to deepen our understanding of the relationship found between HPP and autoimmune diseases to manage these diseases correctly.