Characterising Pain, QoL, Body Composition, Arterial Stiffness, Muscles and Bones in Adult Persons With XLH and Healthy Controls
Characterising Pain, QoL, Body Composition, Arterial Stiffness, Muscles and Bones in Adult Persons With XLH and Healthy Controls
Hereditary hypophosphatemia (XLH) is a rare, inherited disease. Loss-of-function mutation in
 the phosphate regulating gene with homologies to endopeptidases on the X-chromosome (PHEX)
 results in excess fibroblast growth factor 23 (FGF23) production and manifests as rickets in
 children and osteomalacia in adults.

 This study aims to characterize and measure pain, quality of life, muscle function, body
 composition, arterial stiffness, bone mineral density, geometry and microarchitecture in
 patients with XLH compared to age and gender-matched controls.
Hereditary hypophosphatemia (XLH) is a rare, inherited disease. Loss-of-function mutation in
 PHEX results in excess fibroblast growth factor 23 (FGF23) production and manifests as
 rickets in children and osteomalacia in adults. FGF23 is a hormone that reduces renal
 phosphate reabsorption, decreases renal 1α-hydroxylase activity and increases renal
 24-hydroxylase activity. As a consequence, individuals with XLH display hypophosphatemia and
 inadequate levels of 1,25(OH)2D (1). Therefore, conventional medical treatment of XLH aims to
 replace the loss with oral phosphate and activated vitamin D analogues.

 The pain experienced by patients with hypophosphatemic rickets is not well characterized and
 needs to be addressed in order to establish the most optimal pain management in patients with
 XLH. The causes of pain experienced by patients with XLH are numerous: osteomalacia,
 enthesopathy, muscular pain, lower limb deformities, secondary arthrosis, nerve compression,
 and dental abscesses (3).

 Quality of life (QoL) in patients with XLH is only briefly studied (4, 5). A French study
 found significantly decreased QoL among adult patients with XLH compared to patients with
 axial spondylarthritis. Especially enthesopathies were associated with a decreased QoL (4).

 Increased blood levels of FGF23 are not associated with increased risk of cardiovascular
 disease in itself (6-8). However, conventional therapy for patients with XLH may increase
 their cardiovascular risk since complications to the therapy such as nephrocalcinosis and
 hyperparathyroidism are associated with increased cardiovascular risk. Arterial stiffness may
 be a valuable marker of increased cardiovascular risk as it has been able to predict
 cardiovascular disease and mortality in different populations (10, 11). If conventional
 therapy increases the risk of cardiovascular morbidity and mortality, arterial stiffness may
 be increased among patients with XLH which may depend on treatment status (i.e., currently
 treated, currently non-treated, accumulated (years of) treatment and treatment naïve).

 Patients with XLH often complain about muscle fatigue and exhaustion. Muscle function in XLH
 is thought to be compromised by chronic hypophosphatemia, but the effect of XLH on muscle
 function has only been briefly evaluated (12, 13).

 There are no available reports on body composition in XLH adults, but body mass index (BMI)
 in XLH is increased compared to controls (14).

 In previous studies, examinations by high resolution peripheral quantitative computed
 tomography (HRpQCT) have shown significantly larger total bone area in patients with XLH
 compared to individuals without XLH. However, patients with XLH have a reduced number of
 trabeculae and cortical thickness (15). These differences are seen regardless of treatment.
 When receiving conventional treatment, the cortical porosity is significantly elevated
 compared to patients with XLH who receive no treatment (17).

 HYPOTHESES:

 Primary null hypotheses:

 -  There is no difference in pressure pain threshold between groups.

 -  There is no difference in QoL between groups.

 -  There is no difference in arterial stiffness assessed by tonometry between groups.

 -  There is no difference in 24h blood pressure between groups.

 -  There is no difference in muscle strength and function between groups.

 -  There is no difference in body composition between groups.

 -  There is no difference in bone mineral density, geometry, microarchitecture or estimated
 strength between groups.

 -  There is no difference in presence of osteoid-volume, -surface or -thickness, osteoclast
 number, osteoblastic surface, osteoblast morphology or mineralization time between
 groups.

 -  Osteomalacia is not more common in bone biopsies from patients with XLH compared to
 controls.

 Secondary null hypotheses:

 -  There is no difference in arterial stiffness within the XLH group when stratifying for
 treatment status.

 -  There is no difference in 24h blood pressure within the XLH group when stratifying for
 treatment status.

 -  There is no difference in bone mineral density, geometry, microarchitecture or estimated
 strength within the XLH group when stratifying for treatment status.

 MATERIALS AND METHODS

 Study design:

 A cross-sectional study of adult persons with XLH and an age- and gender-matched control
 group.

 Population:

 The investigators plan to include 50 persons with XLH and 50 control persons without
 disturbances in the calcium, vitamin D or phosphate homeostasis, matched by age (+/- 2 years)
 and gender. For women, matching will also be performed on menopausal status.

 EXAMINATIONS

 Questionnaires:

 Pain will be assessed by a bone-specific questionnaire (FACT-BP), a questionnaire for general
 pain (Brief Pain Inventory (BPI)), and one for assessment of neuropathic pain (painDETECT).

 The participant's level of catastrophic thinking will be assessed with the Pain
 Catastrophizing Scale (PCS). Depression and anxiety will be assessed with the Patient Health
 Questionnaire (PHQ-9) and Generalized Anxiety Disorder 7 (GAD-7).

 Quality of Life will be measured by the SF-36 questionnaire (SF36v2).

 Pressure algometry:

 Mechanical pressure pain over the bone will be determined as force (N) per skin area (cm2) by
 a handhold pressure algometer with a 1 cm2 probe (Algometer II, Somedic SenseLab AB) (18).
 The 1 cm2 probe will be directed perpendicularly to the skin and pressure will be applied at
 a constant force increase rate until the participant identifies the pressure as pain and
 presses a button (pressure pain threshold (PPT)). PPT will be measured over the sternum and
 also corresponding to the tibia 5 cm below the patella. The skin between the thumb and the
 index finger of the non-dominant hand will be measured as a control site.

 Biochemistry:

 Blood samples will be collected in a fasting state. The investigators will measure ionized
 calcium, phosphate, magnesium, creatinine, parathyroid hormone (PTH), vitamin D metabolites
 (such as 25OHD; 1,25(OH)2D; 24,25(OH)2D; 1,24,25(OH)3D), vitamin D binding protein, FGF23,
 sclerostin, Klotho, osteopontin, and bone turnover markers (such as CTX, P1NP, osteocalcin
 and bone-specific alkaline phosphatase), and relevant genes for hereditary hypophosphatemia
 (if not previously genetically verified).

 24 hour urine will be collected for measurement of calcium, phosphate, magnesium, creatinin,
 sodium and potassium.

 Blood pressure and arterial stiffness 24-hour blood pressure will be measured using
 Arteriograph24. Arterial stiffness and pulse wave analysis (PWA) will be assessed by
 tonometry using the SphygmoCor system (Xcel; AtCor Medical, Sydney, NSW, Australia).
 Participants treated with betablockers will be excluded from blood pressure and arterial
 stiffness measurements.

 Muscle function The investigators will assess upper and lower extremity strength by handgrip
 strength, elbow flexion and extension, and knee flexion and extension using the adjustable
 dynamometer chair (Good Strength; Metitur Ltd, Finland). To assess physical function, the
 investigators will use the Timed Up and Go (TUG) test, repeated chair rising, repeated weight
 lifting and 6-minutes' walk test (6 MWT).

 Physical examination In order to quantify the degree of malformation of the joints in the
 lower extremities, the smallest distance between knees and between ankles will be measured in
 standing, weight bearing position.

 Joint mobility The investigators will assess joint function in enthesopathy-suspected joints
 (neck/back, hip, knee and ankle) as well as shoulder, elbow, wrist and knee in order to
 discriminate between pain originating from osteomalacia or from enthesopathies.

 Body composition and VFA using DXA Body composition and bone mineral density (Forearm, hip,
 lumbar spine and whole body) will be assessed by dual energy X-ray absorptiometry (DXA)
 (Discovery A scanner; Hologic, USA). Obtaining vertebral fracture assessment (VFA) by DXA
 enables us to exclude reported back pain being due to vertebral compression fractures.

 High-resolution peripheral Quantitative Computed Tomography:

 A HRpQCT bone scan of the dominant distal tibia and radius will be performed using an Xtreme
 CT-scanner (SCANCO Medical AG, Switzerland).

 Bone biopsies:

 Optional bone biopsies will be taken after assessment of pain characteristics. Participation
 in the overall study is allowed without participating in this examination.

 One 7 mm biopsy marked with tetracyclin 14, 13, 4, and 3 days prior will be fixed in ethanol
 and analysed by HRpQCT, Nano-scale and histomorphometry.
Inclusion Criteria:

 -  Understand oral and written Danish

 -  Able to consent

 For XLH only:

 1. genetically verified XLH by detection of a disease-causing mutation in PHEX or a
 positive family history of X-linked hypophosphatemia.

 2. biochemically verified hereditary hypophosphatemia: serum PO4 below normal range and
 low TmPO4/GFR, and/or elevated serum FGF23 and a history of childhood rickets or
 spontaneous endodontic abscesses to exclude acquired hypophosphatemia, e.g.,
 tumor-induced osteomalacia.

 Exclusion Criteria:

 -  P-25OHD < 25 mmol/L*

 -  Severe co-morbidities, which in the opinion of the investigator may have major
 impact on study outcomes. This may include, but is not limited to o poorly
 controlled hyperthyroidism o Paget disease

 o type 1 diabetes mellitus or poorly controlled type 2 diabetes mellitus

 o severe and chronic cardiac, liver, or renal disease

 o Cushing syndrome

 o Rheumatoid arthritis

 o Active pancreatitis

 o Malnutrition

 o Recent prolonged immobility*

 o Active malignancy (including myeloma)

 -  Treatment with

 o Burosumab

 -  Beta-blockers

 -  Oral steroids

 -  For controls only:

 -  disturbances in the calcium or phosphate homeostasis

 -  participants with low 25OHD levels or recent immobility may be
 re-screened for participations 6 months after this has been corrected