Introduction
Materials and methods
Study design
Genetic analysis
Sanger sequencing
MLPA
Clinical evaluation
Biochemical and hormonal assessment
Bone mineral density measurement
Six-point clinical score
Statistical analysis
Results
Genetic variants of ALPL in the series of hypophosphatasemic patients
Patient ID | Exon/Intron | dbSNP* | DNA | Protein | Clinical significance | Haplotype |
---|---|---|---|---|---|---|
1 | Exon 9 | – | c.921del | p.(Ser308Hisfs*58) | Pathogenic | Heterozygosity |
2 | Exon 6 | rs121918019 | c.526G > A | p.(Ala176Thr) | Likely pathogenic, pathogenic | Heterozygosity |
Exon 5 | rs1780316 | c.330 T > C | p.(Ser110Ser) | Likely benign, benign | Heterozygosity | |
Intron 8 | rs1256328 | c.793-31C > T | – | Benign | Homozygosity | |
Exon 12 | rs34605986 | c.1565 T > C | p.(Val522Ala) | Likely benign, benign | Heterozygosity | |
3 | Exon 5 | rs121918011 | c.407G > A | p.(Arg136His) | Pathogenic,uncertain-significance | Heterozygosity |
4 | Exon 5 | rs121918011 | c.407G > A | p.(Arg136His) | Pathogenic,uncertain-significance | Heterozygosity |
5 | Exon 6 | rs121918007 | c.571G > A | p.(Glu191Lys) | Pathogenic-likely, pathogenic | Heterozygosity |
6 | Exon 5 | rs1780316 | c.330 T > C | p.(Ser110Ser) | Likely benign, benign | Homozygosity |
Exon 5 | rs149344982 | c.455G > A | p.(Arg152His) | Likely benign, benign | Heterozygosity | |
Intron 3 | rs1767430 | c.181 + 52C > A | – | Benign | Heterozygosity | |
7 | Exon 5 | rs1780316 | c.330 T > C | p.(Ser110Ser) | Likely benign, benign | Homozygosity |
8 | Exon 5 | rs1780316 | c.330 T > C | p.(Ser110Ser) | Likely benign, benign | Homozygosity |
9 | Exon 5 | rs1780316 | c.330 T > C | p.(Ser110Ser) | Likely benign, benign | Homozygosity |
Exon 7 | rs3200254 | c.787 T > C | p.(Tyr263His) | Benign | Heterozygosity | |
Exon 9 | rs3200255 | c.876A > G | p.(Pro292Pro) | Benign | Heterozygosity | |
Intron 9 | rs74063111 | c.863-7 T > C | – | Benign | Heterozygosity | |
Intron 9 | rs75829132 | c.863-12C > G | – | Benign | Heterozygosity | |
Intron 11 | rs4654760 | c.1309 + 46C > T | – | Benign | Heterozygosity | |
10 | Exon 5 | rs1780316 | c.330 T > C | p.(Ser110Ser) | Likely benign, benign | Homozygosity |
Exon 5 | rs3200254 | c.787 T > C | p.(Tyr263His) | Benign | Heterozygosity | |
Exon 9 | rs3200255 | c.876A > G | p.(Pro292Pro) | Benign | Heterozygosity | |
Intron 9 | rs74063111 | c.863-7 T > C | – | Benign | Heterozygosity | |
Intron 9 | rs75829132 | c.863-12C > G | – | Benign | Heterozygosity | |
11 | Exon 5 | rs1780316 | c.330 T > C | p.(Ser110Ser) | Likely benign, benign | Homozygosity |
Exon 5 | rs149344982 | c.455G > A | p.(Arg152His) | Likely benign, benign | Heterozygosity | |
12 | Exon 7 | rs3200254 | c.787 T > C | p.(Tyr263His) | Benign | Heterozygosity |
Intron 8 | rs2275377 | c.862 + 20G > T | – | Benign | Heterozygosity | |
Intron 8 | rs2275376 | c. 862 + 51G > A | – | Benign | Heterozygosity | |
Intron 8 | rs2275375 | c.862 + 58C > T | – | Benign | Heterozygosity | |
13 | Intron 3 | rs1767430 | c.181 + 52C > A | – | Benign | Heterozygosity |
Exon 5 | rs1780316 | c.330 T > C | p.(Ser110Ser) | Likely benign, benign | Heterozygosity | |
Intron 5 | rs564375559 | c.472 + 12insG | – | Benign | Heterozygosity | |
Exon 7 | rs3200254 | c.787 T > C | p.(Tyr263His) | Benign | Heterozygosity | |
Intron 8 | rs2275377 | c.862 + 20G > T | – | Benign | Heterozygosity | |
Intron 8 | rs2275376 | c. 862 + 51G > A | – | Benign | Heterozygosity | |
Intron 8 | rs2275375 | c.862 + 58C > T | – | Benign | Heterozygosity | |
Exon 9 | rs3200255 | c.876A > G | p.(Pro292Pro) | Benign | Heterozygosity | |
Intron 9 | rs75829132 | c.863-12C > G | – | Benign | Heterozygosity | |
Intron 9 | rs74063111 | c.863-7 T > C | – | Benign | Heterozygosity |
Clinical features in hypophosphatasemic patients according the ALPL genotype
ALPL variants | dbSNP ID | ACMGG/AMP classification* | Patient ID | Patients clinical features | Reported clinical features |
---|---|---|---|---|---|
c.181 + 52C > A | rs1767430 | Benign | 6 | Recurrent abortions, multiple vertebral fractures, hyperkyphosis, premature loss of teeth, hypercalciuria, familiarity for osteoporotic fractures | No report available |
13 | Costal fracture, joint and muscular pain, familiarity for HPP | ||||
c.330T > C, p.(Ser110Ser) | rs1780316 | Benign | 2 | Adrenal cancer, familiarity for HPP | Found in patients with atypical femur fractures and in general population with the same frequency [27] |
6 | Recurrent abortions, multiple vertebral fractures, hyperkyphosis, premature loss of teeth, hypercalciuria, familiarity for osteoporotic fractures | ||||
7 | Pituitary macroadenoma, joint pain, familiarity for osteoporotic fractures | ||||
8 | Multiple vertebral fractures, joint and muscular pain, premature loss of teeth, kidney stones | ||||
9 | Multiple vertebral fractures, seizure, kidney stones, hypercalciuria | ||||
10 | Premature loss of teeth, hypercalciuria, familiarity for HPP | ||||
11 | Breast cancer | ||||
13 | Rib fracture, joint and muscular pain, familiarity for HPP | ||||
c.407G > A, p.(Arg136His) | rs121918011 | Pathogenetic | 3 | Multiple vertebral fractures, premature loss of teeth, kidney stones, goiter | This variant was identified either as single mutation or in compound heterozigosity with other pathogenic ALPL variants in infantile, childhood, adult and odontoHPP (ALPLDB) |
4 | Multiple vertebral fractures, recurrent caries, hyperkyphosis, joint and muscular pain, familiarity for osteoporotic fractures | ||||
c.455G > A, p.(Arg152His) | rs149344982 | Benign | 6 | Recurrent abortions, multiple vertebral fractures, hyperkyphosis, premature loss of teeth, hypercalciuria, familiarity for osteoporotic fractures | Fragility fractures, premature loss of teeth, joint and muscular pain (personal data by Guarnieri V.) |
11 | Breast cancer | ||||
c.472 + 7C > G | rs564375559 | Benign | 13 | Costal fracture, joint and muscular pain, familiarity for HPP | No report available |
c.526G > A, p.(Ala176Thr) | rs121918019 | Pathogenetic | 2 | Adrenal cancer, familiarity for HPP | This variant was identified either as single mutation or in compound heterozigosity with other pathogenic ALPL variants in infantile, childhood, adult and odontoHPP (ALPLDB) |
c.571G > A, p.(Glu191Lys) | rs121918007 | Pathogenetic | 5 | Multiple vertebral fractures, clavicular fracture, recurrent caries | This variant was identified either as single mutation or in compound heterozigosity with other pathogenic ALPL variants in infantile, childhood, adult and odontoHPP (ALPLDB) |
c.787T > C, p.(Tyr263His) | rs3200254 | Benign | 9 | Multiple vertebral fractures, seizure, kidney stones, hypercalciuria | Found in patients with atypical femur fractures and in general population with the same frequency [27] |
10 | Premature loss of teeth, hypercalciuria, familiarity for HPP | ||||
12 | Autoimmune hypothyroidism, rib fractures, kidney stones, hypercalciuria | ||||
13 | Costal fracture, joint and muscular pain, familiarity for HPP | ||||
c.793-31C > T | rs1256328 | Benign | 2 | Adrenal cancer, familiarity for HPP | |
c.862 + 20G > T | rs2275377 | Benign | 12 | Autoimmune hypothyroidism, rib fractures, kidney stones, hypercalciuria | Found in linkage disequilibrium in patients with mild HPP (childhood and adult HPP, odontoHPP, and perinatal benign HPP) [9] |
13 | Rib fracture, joint and muscular pain, familiarity for HPP | ||||
c.862 + 51G > A | rs2275376 | Benign | 12 | Autoimmune hypothyroidism, rib fractures, kidney stones, hypercalciuria | Found in linkage disequilibrium in patients with mild HPP (childhood and adult HPP, odontoHPP, and perinatal benign HPP) [9] |
13 | Rib fracture, joint and muscular pain, familiarity for HPP | ||||
c.862 + 58C > T | rs2275375 | Benign | 12 | Autoimmune hypothyroidism, rib fractures, kidney stones, hypercalciuria | Found in linkage disequilibrium in patients with mild HPP (childhood and adult HPP, odontoHPP, and perinatal benign HPP) [9] |
13 | Rib fracture, joint and muscular pain, familiarity for HPP | ||||
c.863-7T > C | rs74063111 | Benign | 9 | Multiple vertebral fractures, seizure, kidney stones, hypercalciuria | No report available |
10 | Premature loss of teeth, hypercalciuria, familiarity for HPP | ||||
13 | Costal fracture, joint and muscular pain, familiarity for HPP | ||||
c.863-12C > G | rs75829132 | Benign | 9 | Multiple vertebral fractures, seizure, kidney stones, hypercalciuria | No report available |
10 | Premature loss of teeth, hypercalciuria, familiarity for HPP | ||||
13 | Costal fracture, joint and muscular pain, familiarity for HPP | ||||
c.876A > G, p.(Pro292Pro) | rs3200255 | Benign | 9 | Multiple vertebral fractures, seizure, kidney stones, hypercalciuria | Found in patients with atypical femur fractures and in general population with the same frequency [27] |
10 | Premature loss of teeth, hypercalciuria, familiarity for HPP | ||||
13 | Costal fracture, joint and muscular pain, familiarity for HPP | ||||
c.921delG p.(Ser308Hisfs*58) | NR | Pathogenetic | 1 | Breast cancer, vertebral, wrist and humerus fractures, recurrent caries, scoliosis, familiarity for osteoporotic fractures | No report available |
c.1309 + 46C > T | rs4654760 | Benign | 9 | Multiple vertebral fractures, seizure, kidney stones, hypercalciuria | No report available |
c.1565T > C, p.(Val522Ala) | rs34605986 | Benign | 2 | Adrenal cancer, familiarity for HPP | Found in patients with atypical femur fractures and in general population with the same frequency [27] |
Parameters | Pathogenic ALPL variants | Benign ALPL variants | Wild-type ALPL alleles | P |
---|---|---|---|---|
Age (years) | 58.8 ± 3.3 | 59.1 ± 4.5 | 53.4 ± 3.8 | 0.535 |
BMI (kg/m2) | 25.8 ± 0.9 | 21.7 ± 1.8 | 23.7 ± 2.1 | 0.453 |
Serum total ALP (U/L) | 26.1 ± 2.3 | 32.3 ± 2.5 | 30.8 ± 2.3 | 0.274 |
Serum CTX (ng/mL) | 0.541 ± 0.088 | 0.482 ± 0.081 | 0.285 ± 0.054 | 0.051 |
Serum total calcium (mg/dL) | 9.2 ± 0.2 | 9.3 ± 0.1 | 9.4 ± 0.1 | 0.723 |
Serum phosphate (mg/dL) | 3.8 ± 0.2 | 3.4 ± 0.2 | 3.3 ± 0.2 | 0.237 |
Serum 25OHD (ng/mL) | 29.6 ± 5.2 | 39.0 ± 3.3 | 42.8 ± 3.8 | 0.112 |
Serum 25OHD < 30 ng/mL, n (%) | 3 (60%) | 1 (12.5%) | 1 (11%) | 0.080 |
Plasma PTH (pg/mL) | 55.3 ± 8.0 | 41.2 ± 8.5 | 56.3 ± 7.8 | 0.205 |
UCa (mg/24 h) | 186.4 ± 25.5 | 212.3 ± 21.8 | 154.4 ± 11.0 | 0.101 |
UCa (mg/kg/24 h) | 2.7 ± 0.3 | 3.8 ± 0.5 | 2.6 ± 0.2 | 0.048 |
L1-L4 T-score | − 1.96 ± 0.74 | − 1.94 ± 0.78 | − 1.54 ± 0.37 | 0.849 |
Femoral neck T-score | − 2.14 ± 0.38 | − 1.88 ± 0.27 | − 1.50 ± 0.36 | 0.453 |
Total hip T-score | − 1.33 ± 0.59 | − 1.41 ± 0.29 | − 1.27 ± 0.30 | 0.950 |
DXA osteoporosis, n (%) | 2 (40%) | 4 (50%) | 4 (44%) | 0.937 |
Urinary PEA (mmol/mol creatinine) | 5.8 ± 0.3 | 3.1 ± 0.7 | 2.3 ± 0.6 | 0.005 |
Serum PLP (µg/L) | 30.5 ± 13.6 | 11.0 ± 0.9 | 10.6 ± 2.0 | 0.050 |
Risk factors | Pathogenic ALPL variants | Benign ALPL variants | Wild-type ALPL alleles | P value |
---|---|---|---|---|
Dental anomalies (%) | 4/1 (80)* | 3/5 (38) | 0/9 (0) | 0.023 |
Neurologic and musculoskeletal symptoms (%) | 2/3 (40) | 4/4 (50)§ | 0/9 (0) | 0.027 |
Mineral metabolism anomalies (%) | 1/4 (20) | 5/3 (63)# | 0/9 (0) | 0.014 |
Familiarity for osteoporosis and/or fragility fractures (%) | 3/2 (60) | 5/3 (71) | 1/8 (10) | 0.061 |
All fractures (%) | 4/1 (80)°° | 5/3 (57)° | 1/8 (20) | 0.022 |
Vertebral fractures (%) | 4/1 (80) | 3/5 (37) | 1/8 (20) | 0.037 |
Increased urine PEA and/or serum PLP | 5/0 (100) | 6/2 (75) | 5/4 (55) | 0.199 |
Six-points score (mean ± SEM) | 4.0 ± 0.6 | 3.4 ± 0.5 | 0.8 ± 0.2 | 0.0001 |
Tamoxifen treatment (%) | 0/5 (0)$$ | 1/7 (13)$ | 6/3 (67) | 0.013 |