Genotype-Phenotype Associations in 72 Adults with Suspected ALPL-Associated Hypophosphatasia

Abstract

Hypophosphatasia (HPP) is a rare inborn error of metabolism due to a decreased activity of tissue nonspecific alkaline phosphatase (TNSALP). As the onset and severity of HPP are heterogenous, it can be challenging to determine the pathogenicity of detected rare ALPL variants in symptomatic patients. We aimed to characterize patients with rare ALPL variants to propose which patients can be diagnosed with adult HPP. We included 72 patients with (1) clinical symptoms of adult HPP or positive family history and (2) low TNSALP activity and/or high pyridoxal 5'-phosphate (PLP) levels, who underwent ALPL gene sequencing. The patients were analyzed and divided into three groups depending on ALPL variant pathogenicity according to the classification of the American College of Medical Genetics and Genomics (ACMG). Reported pathogenic (n = 34 patients), rare (n = 17) and common (n = 21) ALPL variants only were found. Muscular complaints were the most frequent symptoms (> 80%), followed by bone affection (> 50%). Tooth involvement was significantly more common in patients with pathogenic or rare ALPL variants. Seven rare variants could be classified as likely pathogenic (ACMG class 4) of which five have not yet been described. Inconclusive genetic findings and less specific symptoms make diagnosis difficult in cases where adult HPP is not obvious. As not every pathogenic or rare ALPL variant leads to a manifestation of HPP, only patients with bone complications and at least one additional complication concerning teeth, muscle, central nervous and mental system, repeated low TNSALP activity and high PLP levels should be diagnosed as adult HPP if rare ALPL gene variants of ACMG class 4 or higher support the diagnosis.

Keywords: ALP; Alkaline phosphatase; HPP; PLP; Pyridoxal 5′-phosphate; TNSALP.

Fig. 1

Flowchart with the clinical workflow for patients with signs and symptoms of HPP or a family history of HPP. After detection of laboratory abnormalities with low tissue nonspecific alkaline phosphatase (TNSALP) or bone-specific alkaline phosphatase (BAP) activity and high pyridoxal 5′-phosphate (PLP), ALPL gene sequencing is routinely performed. Three groups of ALPL variants may be distinguished: (1) Reported variants, that are listed in the ALPL gene mutations database by the SESEP laboratory [2] and classified as pathogenic variants, (2) rare variants as well as variants of uncertain significance (VUS), and (3) reported common variants representing ALPL polymorphisms without pathogenicity. If a reported pathogenic variant is found, the diagnosis of HPP is supported by the genetic test result. In the presence of a rare variant, the diagnosis must be strictly related to the clinical presentation of the patient, and diagnosing HPP can be challenging in mild forms of adult HPP. The detection of a rare variant with unknown pathogenicity does not support the physician in diagnosing mild adult HPP. Furthermore, it is even more difficult for the physician to assess the presence of HPP if only reported common variants are found in a symptomatic patient. If necessary, more extensive genetic investigations (quantitative multiplex PCR of short fluorescent fragments, exome or genome analysis) could provide additional diagnostic certainty in the future, as this allows the detection of large deletions in the ALPL gene and the exclusion of differential diagnoses

Fig. 2

Representative possible clinical complications of adult HPP. a MR images (coronal and sagittal, PD-weighted, fat-saturated, turbo spin echo) of a stress fracture of the distal tibia (white arrows), b tooth hypomineralization and caries, c muscle weakness measured by grip force with digital hand dynamometry. d Muscle symptoms were leading in all three patient groups with pathogenic, rare, or common ALPL variants. Dental affection was significantly more frequent in patients with pathogenic or rare variants compared to patients with common variants (*p < 0.05)

Fig. 3

Frequency and laboratory values of patients with reported pathogenic, rare, or common variants in the ALPL gene in the overall cohort. a All reported pathogenic variants are listed in the hypophosphatasia online database [2] and classified as pathogenic (class 5) according to ACMG criteria. For patients with compound-heterozygous pathogenic ALPL variants both variants are listed (c.1001G > A (p.Gly334Asp) + c.283G > A (p.Val95Met)). For patients with one pathogenic and a second VUS (class 3) or likely pathogenic variant, only pathogenic variants are displayed (c.571G > A (p.Glu191Lys) + c.1018C > T (p.His340Tyr), c.542C > T (p.Ser181Leu) + c.818C > T (p.Thr273Met)). ^aCAGGGGAinsT, *Pathogenic variant, which has so far only been described as pathogenic in combination with a second mutation. b Rare variants with allele frequency < 0.01 according to gnomAD (class 3 and 4). c Reported common variants classified as benign (class 1) are depicted in the third bar chart. d–f Median TNSALP, BAP and PLP levels of patients with pathogenic, rare, and common variants. Pathogenic variants were associated with significantly lower TNSALP (***p < 0.001) and BAP (*p = 0.01) compared to patients with common variants. Patients with rare variants showed significantly lower BAP (p = 0.043) and borderline significant lower TNSALP (p = 0.051) compared to patients with common variants. TNSALP, BAP and PLP did not differ significantly between patients with pathogenic and rare variants. g–i In 3, 9 and 3 of 34 patients with pathogenic variants, TNSALP and BAP activity as well as PLP levels were in the normal range compared to gender- and age-specific reference values. j–l In 6, 4 and 2 of 17 patients with rare variants, TNSALP, BAP and PLP levels were in the normal range compared to gender- and age-specific reference values

Fig. 4

Rare variants with allele frequency (AF) in the European (Non-Finnish) population, in silico prediction results (CADD, REVEL), laboratory values and clinically affected organ systems. Bone (stress fractures, pseudarthrosis, bone marrow edema, low-traumatic fractures, low bone mineral density (T-Score ≤ −2.5), osteonecrosis, scoliosis), tooth (hypomineralization, tooth extraction, early tooth loss, malposition of teeth, periodontitis, caries), muscle (myalgia, tendinopathy, reduced grip force, fatigue) and neurological/psychiatric symptoms (frequent cephalgia, migraine, depression, use of antidepressants). The horizontal boxes indicate the frequency of symptoms for each variant in percentages (full box = 100%, empty box = 0%). The colored circles indicate the proportion of cases for each variant in which alkaline phosphatase (ALP), bone-specific alkaline phosphatase (BAP) and pyridoxal 5′-phosphate (PLP) are pathologic (red: low ALP/BAP, high PLP) or within the lower/upper normal range (green) compared to gender- and age-specific reference values. Adult HPP was diagnosed, if ALP or BAP activity was repeatedly below the reference range or around the lower limit value together with elevated PLP, bone complications and at least one further complication concerning teeth, muscle, central nervous and mental system supported by at least one rare ALPL variant (ACMG class 4 or higher)