The clinical characteristics of the three adult patients with APS1 (female P1: 38 years; male P2: 47 years; male P3: 25 years) are shown in Table 1. As signs of osteoporosis, P1 had experienced a vertebral compression fracture, whereas P2 and P3 had osteoporotic BMD. P3 had experienced five fractures due to high-energy accidents; all the fractures had been treated successfully with immobilization 4–12 years before bone biopsy. None of the patients were treated with bisphosphonates or other osteoporosis medications prior to biopsy.

All patients had biallelic AIRE mutations and 7–10 disease manifestations. P1 and P2 had both hypoparathyroidism and adrenal insufficiency, while P3 did not have hypoparathyroidism and needed hydrocortisone substitution only in the case of stress for early adrenal insufficiency. P1 and P3 had hypergonadotropic hypogonadism treated with contraceptive pills containing ethinylestradiol and with testosterone, respectively. P3 had stable renal tubular acidosis treated adequately with sodium substitution and sodium bicarbonate. Exocrine pancreas insufficiency was treated with enzyme substitution at meals in P3. Due to intestinal dysfunction, P1 had used lactobacillus supplement for years and P2 avoided dairy products.

P1 and P2 had short stature, whereas P3 was under-weighted. P1 and P2 had ionized calcium level slightly below normal range, but within the target range for patients with hypoparathyroidism. Whole body BMD T-scores were + 1.2, − 2.8, and − 3.5 for P1, P2, and P3, respectively.

In bone biopsies, bone volume (BV/TV) was in the normal range for all patients (Table 2, Fig. 1). Nevertheless, it was in the lower range for P1 and P3, which was mainly due to reduced trabecular thickness, but partly reversed by an increase in trabecular number.

Typical microarchitectures of iliac crest specimens under light microscopy (1.1–1.2; 2.1–2.2; 3.1–3.2) and fluorescence microscopy (1.3; 2.3; 3.3) are exemplified from patients 1, 2 and 3 with APS1. The blue arrows indicated the peri-trabecular fibrosis and the double tetracycline labels seen in cortical bone were shown by yellow arrows in patient 1. The magnified images (highlighted by red rectangle) demonstrated the normal double labeling in cancellous bone of patient 2 and patient 3. Masson Goldner trichrome stain

In P1, the amount of osteoid was low; active osteoblasts and tetracycline double labels were seen only on cortical bone. Bone resorption was increased (Table 2), and there was peritrabecular fibrosis of bone marrow (Fig. 1). Dynamic parameters in cancellous bone could be measured in P2 and P3. In P2, dynamic parameters were within the normal range. In P3, no osteoclast was seen, and increased bone turnover rate (BFR/BV + 4.53 SD) associated with an increase in osteoid tissue was observed (Fig. 1). Double labels were seen in P3 ruling out the presence of pronounced osteomalacia. However, focal mineralization defects were identified as the presence of “osteoid islands” surrounded by mineralized bone (online resource).

The three samples showed different bone matrix mineralization characteristics. Figure 2A shows the trabecular BMDD curves obtained for the three patients, as well as the adult reference curve for comparison [10]; online resource shows the resulting BMDD parameters for trabecular and cortical compartment. P1 showed a slight hypermineralization for the trabecular and cortical compartment as well as a decrease in the width of the BMDD (CaMeanTrab + 1.75 SD, CaWidthTrab − 2.04 SD, CaMeanCort + 1.59 SD, CaWidthCort − 1.53 SD) denoting a more homogenous mineralization compared to reference. In contrast, the trabecular BMDDs from P2 and P3 showed a considerable broadening of the curve. In P3, it was accompanied with a pronounced hypomineralization reflected in a large decrease in CaMean and CaPeak and a large increase in CaLow, respectively. The findings of qBEI were also reflected in the results from histomorphometry. The amount of osteoid volume and osteoid surface (Table 2) were consistent with the mineralization results, as a high turnover rate pointed towards a reduced degree of mineralization. Furthermore, similar with the histology pronounced focal mineralization defects could be spotted in P2 and P3 (online resource).

A shows the BMDD curves for trabecular bone of patients 1, 2, and 3, respectively. The black dashed line corresponds to the adult reference BMDD curve, the grey band depicts the corresponding standard deviation[10]. B show the results of the OLS analysis for OLS-density and OLS-area for cortical and trabecular compartments. Data are compared against results from 2 healthy women aged 36 and 42 years[12]

The OLS-density and OLS-area were similar to reference samples obtained from two healthy adults in both cortical and trabecular bone (Fig. 2B).