Bone Reports 15 (2021) 101101 Contents lists available at ScienceDirect Bone Reports journal homepage: www.elsevier.com/locate/bonr Biochemical, clinical and genetic characteristics in adults with persistent hypophosphatasaemia; Data from an endocrinological outpatient clinic in Denmark Nicola Hepp a,*, Anja Lisbeth Frederiksen b,c, Morten Duno d, Jakob Præst Holm e, Niklas Rye Jørgensen f,g, Jens-Erik Beck Jensen a,g a Dept. of Endocrinology, Hvidovre University Hospital Copenhagen, Kettegaard Alle 30, 2650 Hvidovre, Denmark b Dept. of Clinical Genetics, Aalborg University Hospital, Ladegaardsgade 5, 9000 Aalborg C, Denmark c Dept. of Clinical Research, Aalborg University, Fredrik Bajers Vej 7K, 9220 Aalborg Ø, Denmark d Dept. of Clinical Genetics, University Hospital Copenhagen Rigshospitalet, Blegdamsvej 9, 2100 Copenhagen, Denmark e Department of Endocrinology, Copenhagen University Hospital Herlev, Borgmester Ib Juuls Vej 1, 2730 Herlev, Denmark f Dept. of Clinical Biochemistry, Rigshospitalet, Valdemar Hansens Vej 13, 2600 Glostrup, Denmark g Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3 B, 2200 Copenhagen, Denmark ARTICLE INFO ABSTRACT Keywords: Background: Hypophosphatasia (HPP) is an inborn disease caused by pathogenic variants in ALPL. Low levels of Alkaline phosphatase alkaline phosphatase (ALP) are a biochemical hallmark of the disease. Scarce knowledge about the prevalence of ALPL HPP in Scandinavia exists, and the variable clinical presentations make diagnostics challenging. The aim of this Hypophosphatasia study was to investigate the prevalence of ALPL variants as well as the clinical and biochemical features among Osteoporosis adults with endocrinological diagnoses and persistent hypophosphatasaemia. Bisphosphonates Methods: A biochemical database containing ALP measurements of 26,121 individuals was reviewed to identify adults above 18 years of age with persistently low levels of ALP beneath range (≤ 35 ± 2.7 U/L). ALPL genetic testing, biochemical evaluations and assessment of clinical features by a systematic questionnaire among included patients, were performed. Results: Among 24 participants, thirteen subjects (54.2%) revealed a disease-causing variant in ALPL and re­ ported mild clinical features of HPP, of which musculoskeletal pain was the most frequently reported (n = 9). The variant c. 571G > A; p.(Glu191Lys) was identified in six subjects, and an unreported missense variant (c.1019A > C; p.(His340Pro)) as well as a deletion of exon 2 were detected by genetic screening. Biochemical analyses showed no significantdifferences in ALP (p = 0.059), the bone specificalkaline phosphatase (BALP) (p = 0.056) 0 and pyridoxal-5 -phosphate (PLP) (p = 0.085) between patients with an ALPL variant and negative genetic screening. Patients with a variant in ALPL had significantlyhigher PLP levels than healthy controls (p = 0.002). We observed normal ALP activity in some patients classifiedas mild HPP, and slightly increased levels of PLP in two subjects with normal genetic screening and four healthy controls. Among 51 patients with persistent hypophosphatasaemia, fifteen subjects (29.4%) received antiresorptive treatment. Two patients with unrecog­ nized HPP were treated with bisphosphonates and did not show complications due to the treatment. Conclusions: Pathogenic variants in ALPL are common among patients with endocrinological diagnoses and low ALP. Regarding diagnostics, genetic testing is necessary to identify mild HPP due to fluctuating biochemical findings. Antiresorptive treatment is a frequent reason for hypophosphatasaemia and effects of these agents in adults with a variant in ALPL and osteoporosis remain unclear and require further studies. * Corresponding author. E-mail addresses: [email protected] (N. Hepp), [email protected] (A.L. Frederiksen), [email protected] (M. Duno), Niklas.Rye. [email protected] (N. Rye Jørgensen), [email protected] (J.-E. Beck Jensen). https://doi.org/10.1016/j.bonr.2021.101101 Received 19 April 2021; Received in revised form 18 June 2021; Accepted 23 June 2021 Available online 28 June 2021 2352-1872/© 2021 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). N. Hepp et al. Bone Reports 15 (2021) 101101 1. Introduction at least two ALP measurements ≤35 U/L and none ≥35 (± 2.7) U/L, were invited to participate in the study by letter. Patients, who returned Hypophosphatasia (HPP), an inherited metabolic disease character­ a signed reply letter and agreed to receive further information were ized by deficiency of the enzyme alkaline phosphatase (ALP) is caused contacted by telephone. The study included an information conversa­ by pathogenic variants in the Tissue Nonspecific Alkaline Phosphatase tion, a screening visit and a main study visit, where we took blood (TNSALP) encoding gene (ALPL) (Millan´ and Whyte, 2016; Weiss et al., samples for genetic screening, performed a physical examination and a 1988). questionnaire survey. Results of genetic screening where given to the The HPP presents a wide clinical spectrum including both severe participants at a closing conversation with genetic counselling. recessive inherited prenatal and dominant inherited milder forms, Exclusion criteria included renal failure (eGFR ≤40 mL/min), cur­ diagnosed in childhood and adults caused by homozygous/compound rent malignant disorders, Cushing’s disease, manifest and untreated heterozygous or heterozygous pathogenic ALPL variants, respectively hypothyroidism (T4 below normal range) ≥ 6 months (at screening) and (Hofmann et al., 2013). In adults, HPP may debut as fragility fractures pregnancy. All participants provided oral and written informed consent. with delayed healing and dental complications including premature loss The study was carried out in accordance with the Helsinki Declaration of permanent teeth and abnormal formation of cementum and enamel and was approved by the regional ethical committee (Journal-nr.: H- (Berkseth et al., 2013; Mori et al., 2016; Schmidt et al., 2017). 20011193) and the data protection agency (j.nr.: AHH-2017-076; I-Suite Furthermore, the patients report unspecificsymptoms including muscle nr.: 05857). weakness, impaired daily physical activity and chronic pain related to muscles, joints and bones (Hogler¨ et al., 2019; Schmidt et al., 2017; 2.2. Biochemical parameters Weber et al., 2016). The maximal prevalence of mild HPP has recently been estimated to be 1/508 in the European population (Mornet et al., Measurements of ALP activity were performed on a Vitros 5.1 2020). In contrast, only few patients have until now been diagnosed analyzer (Ortho Clinical Diagnostics, Inc., USA) from February 2004 to with HPP in Denmark. May 2010. From May 2010 to date, ALP has been analyzed on the Persistently low levels of ALP represents a characteristically COBAS 8000 from Roche Diagnostics GmbH, Mannheim, Germany. In­ 0 biochemical finding in HPP and pyridoxal-5 -phosphate (PLP), a sub­ ternal laboratory data showed an acceptable comparison between the strate of TNSALP can be elevated (Whyte, 2017) while identificationof two systems. pathogenic variant(s) in ALPL verifies the clinical diagnosis (Mentrup After overnight fasting, blood samples for biochemical analysis were et al., 2017). taken at the screening visit between 8.00 and 10.00 a.m. Participants Milder HPP may however, be unrecognized or misdiagnosed because had paused all supplements, containing vitamin B6 and calcium, for at of the non-specificsymptoms or unawareness of low ALP (Hogler¨ et al., least two weeks. PLP was analyzed by high pressure liquid chromatog­ 2019; Maman et al., 2016). Due to a history of low energy fractures or/ raphy (HPLC) analysis (Chromosystems Instruments and Chemical and low bone mineral density (BMD), patients with unrecognized HPP GmbH, Germany). Batch analyses of PLP were conducted for the ALPL are in risk to be diagnosed with osteoporosis. Case reports indicate that groups and the control groups, respectively. BALP was batch analyzed bisphosphonates (BPs), the most common used drug for osteoporosis, and measured by an iSYS automated analyzer using an immune-based may increase fracture risk and provoke atypical femoral fractures (AFFs) chemiluminescence assay (Immunodiagnostic Systems, Tyne and in patients with osteoporosis and unrecognized HPP (Sum et al., 2013; Wear, UK). Malabu et al., 2019; Sutton et al., 2012). These reports have led to the assumption, that BPs are contraindicated in HPP (Marini and Brandi, 2.3. Genetic analyses 2017; Shapiro and Lewiecki, 2017). In addition, BPs and other antiresorptive drugs suppress bone turn­ Sanger sequencing was performed to screen the coding and intron over and may decrease levels of ALP and the bone-specificALP (BALP), flankingsequences of ALPL (NM_000478.5). DNA samples, in which no an isoform of ALP and marker for bone formation used to monitor variant was identified by Sanger sequencing, were subsequently antiresorptive treatment (Coburn et al., 1998; Morey et al., 2018; Sar­ analyzed by Multiplex Ligation-dependent Probe Amplification(MLPA) ioglu et al., 2006). Consequently, the interpretation of low ALP and thus to identify any larger deletions or duplications. The MLPA was con­ the recognition of HPP remains challenging in patients with secondary ducted according to the manufacturer’s instructions (SALSA MLPA reasons for low ALP including