*2.3.3 Biochemical features and diagnostic biomarkers*

The key biochemical features of HP2 are elevated plasma and urinary levels of proline (about 10–15 folds higher in plasma) and P5C. A combination of both biomarkers is diagnostic of HP2 and distinguishes it from hyperprolinemia type 1 [119, 120]. Walker and Mills [121] identified a new metabolite,

#### **Figure 7.**

*L-Proline metabolic pathway. In HP2, inactivation of P5CD causes accumulation of the upstream metabolite P5C (red arrows). P5C spontaneously condenses with the enzymatic cofactor PLP leading to the formation of inactive adducts and depletion of the cofactor. GSA: glutamic-gamma-semialdehyde, ORN: ornithine, NAD(P): nicotinamide adenine dinucleotide (phosphate), POX: proline oxidase, P5CR: P5C reductase, P5C: pyrroline 5-carboxylate, OAT: ornithine aminotransferase, P5CD: P5C dehydrogenase, P5CS: P5C synthase. (Based on [63, 113]).*

N-(pyrrole-2-carboxyl) glycine, that accumulated in urine of HP2 subject. They subsequently confirmed the presence of this compound in another 4 patients and suggested its use as a diagnostic biomarker for HP2. Other metabolic alterations reported in HP2 patients include increased plasma concentrations of lactate [116, 119], glycine [115, 120], ornithine [120], and alanine [119] and urinary xanthurenic acid; probably secondary to PLP deficiency [59]. VitB6 was previously analyzed in 5 HP2 patients [59, 116, 119] and found to be decreased in 3 patients and at low normal levels in the other two.

#### *2.3.4 Treatment and its outcome*

VitB6 supplementation has been used to treat HP2 associated seizures with variable response. Most of the case studies reported effective control of seizures with vitB6, either alone or in conjugation with anti-seizure medications [59, 114, 116], while few described irresponsiveness to vitB6 therapy [119]. Van de Ven et al. [119] assessed the long-term clinical outcome in 4 HP2 patients treated with vitB6 and/or anti-seizure medications. Seizures resolved spontaneously in 3 patients by the age of 12–18 years, however, neurobehavioral problems were persistent in most patients despite therapy. The clinical course was non-progressive and did not correlate with the vitB6 dose and vitB6 therapy [119].

#### **2.4 Hypophosphatasia**

#### *2.4.1 Disease mechanism*

Hypophosphatasia (HPP) results from autosomal recessive or dominant mutations affecting *ALPL*, the gene encoding tissue nonspecific alkaline phosphatase (TNSALP). TNSALP is an ecto-enzyme that is highly expressed in bone, liver, kidney and developing teeth [122, 123]. The enzyme catalyzes extracellular dephosphorylation of multiple substrates including inorganic pyrophosphate (PPi), phosphoethanolamine (PEA), and PLP (**Figure 3**) [122, 124]. On the osteoblast membrane, TNSALP hydrolyzes PPi into inorganic phosphate (Pi). Together with calcium ions (Ca2+), Pi is required for the synthesis of hydroxyapatite (HA) which is the major inorganic constituent of bones and teeth. In HPP, TNSALP deficiency leads to extracellular accumulation of PPi which impairs the formation of HA and proper bone mineralization leading to an array of skeletal abnormalities [122, 123]. TNSALP is also required for the extracellular hydrolysis of PLP to PL to facilitate its entry into the cell which explains the occurrence of intracellular PLP deficiency and vitB6-dependent seizures in some forms of HPP [123, 124].

#### *2.4.2 Clinical features*

There is a remarkable heterogeneity in the clinical presentation of HPP and 5 principal clinical types have been recognized based on skeletal disease features and age of onset. In order of escalating severity, these types are "odonto", "adult", "childhood", "infantile", and "perinatal" HPP [124, 125]. The severe forms (infantile and perinatal) show autosomal recessive inheritance, while in the milder forms both autosomal dominant or recessive inheritance has been described [124, 126]. Defective mentalization of bone and/or teeth is the clinical hallmark feature of HPP in all of these types [127]. Seizures are the most well described extra-skeletal feature of HPP and are exclusively observed in the infantile and perinatal types [128]. According to a recent metanalysis [128], seizures occurred in about 20% of patients with pediatric-onset HPP.

#### *Vitamin B6 and Related Inborn Errors of Metabolism DOI: http://dx.doi.org/10.5772/intechopen.99751*

Odonto-HPP is the mildest form and can manifest at any age. It involves minor dental problems like premature shedding of deciduous teeth without any other symptoms [123, 124]. Adult HPP typically manifest during middle age or later and can cause debilitating symptoms like osteomalacia leading to bone fractures, chondrocalcinosis, musculoskeletal pain and loss of dentition. Some patients also suffer from pseudogout due to increased extracellular concentrations of PPi [123, 126]. Childhood HPP presents after the age of 6 months and common features include rickets and premature loss of deciduous teeth. Severe forms are also associated with muscle weakness causing delay in walking and abnormal gait [125]. Infantile HPP is a severe type and can lead to death in about 50% of affected infants [123]. It is diagnosed before 6 months of age and features delayed postnatal development, failure to thrive, hypotonia along with rachitic deformities [125]. Hypercalcemia and hypercalciuria are frequently seen and may lead to renal failure [126]. In rapidly progressive cases, rickets causes thoracic deformity and death may ensue due to respiratory insufficiency [125, 129]. VitB6-dependent seizures may develop, sometimes preceding the skeletal features, and usually predict a fatal outcome [123, 125]. Perinatal HPP is the most severe type in which the symptoms start *in utero* or at birth and almost always lead to lethal outcome. Skeletal hypomineralization is profound and causes deformities such as caput membranaceum, wide fontanels and short limb dwarfism [123, 125, 130]. Chest malformation followed by pulmonary compromise is also a common fatal consequence of the rachitic disease [126]. Additional features described in this extreme form of HPP comprise vitB6 dependent seizures, apnea, irritability, myelophthisic anemia and intracranial hemorrhage [123].

## *2.4.3 Biochemical features and diagnostic biomarkers*

HPP can be diagnosed by the presence of pathognomonic skeletal radiographic changes along with characteristic biochemical features. The most commonly used biochemical marker for HPP is low serum alkaline phosphatase activity which consistently observed in all forms of HPP [126]. Other reported biochemical findings in HPP include increased levels of TNSALP substrates PPi and PEA in urine, elevated Pi in plasma, hypercalciuria and/or hypercalcemia and high urinary levels of phosphoserine [6, 123, 124, 126]. These features can only be used to support the diagnosis of HPP because they may not be present in all HPP forms and are sometimes observed in other skeletal diseases. A more sensitive and specific biomarker for HPP is elevated serum levels of PLP, which has been detected even in the mildest form of HPP (odonto-HPP) and the degree of PLP elevation seems to correlate with disease severity [123, 131].

## *2.4.4 Treatment and its outcome*

HPP-related seizures are usually responsive to PN supplementation [56, 129]. Effective treatment against the skeletal manifestations was lacking until the advent of Asfotase alfa, an enzyme-replacement therapy that was approved in 2015 [124, 126]. Asfotase alfa is recombinant, fusion protein consisting of the catalytic ectodomain of human TNSALP, the Fc fragment of human immunoglobulin G1 (IgG1) and a deca-aspartate motif for bone targeting [123, 131, 132]. Clinical trials have demonstrated the long-term safety and efficacy of Asfotase alfa in preventing life-threatening complications of HPP [123, 132, 133]. HPP patients, including those with severe forms, treated with Asfotase alfa showed marked improvements in all clinical aspects (radiography, pulmonary, neurodevelopmental and motor functions) along with resolution of pain and disability

[123, 126, 133]. At the biochemical level, Asfotase alfa therapy was associated with normalization of plasma levels of PPi and PLP [133].

### **2.5 PLPHP deficiency**

#### *2.5.1 Disease mechanism*

PLPHP deficiency is the latest addition to B6EEs that is caused by recessive mutations in *PLPBP*, a gene previosly known as proline synthetase co-transcribed homolog (*PROSC*) [7]. The product of this gene, known as PLP homeostasis protein (PLPHP), belongs to a highly conserved family of proteins known to bind PLP. The function of these PLP-binding proteins in humans as well as other species is poorly understood. Their structures have remarkable similarity with a bacterial enzyme known as alanine racemase [134]. An insight into the function of this protein came from ananlysis of samples from PLPHP-deficient pateints which showed a widely deranged vitB6 vitamer profile. It has therefore been suggeseted that this protein plays an imprtant role in vitB6 homeostasis [7, 68]. However, the exact mechanism of how PLPHP dysfunction disrupts PLP homeostasis and leads to the observed epileptic encephalopathy is still unknown. Darin et al. [7] hypothesized that PLPHP is a PLP-carrier that protects the reactive cofactor from binding to other cellular molecules, shields it from degradative enzymes like phosphatases and securely delivers it to PLP-dependent enzymes.

#### *2.5.2 Clinical features*

The general clinical picture of PLPHP deficiency remarkably overlaps with that of ALDH7A1 deficiency and PNPO deficiency which is dominated by pharmacoresistant seizures that respond to vitB6 treatment. Seizures typically manifest during the first week of life [7, 67, 68, 135] with possible prenatal onset in some cases [68] and a recent report of late onset at 14 months of age [136]. Johnstone et al. [68] reported two patients who presented with fatal mitochondrial encephalopathy and a patient with unique movement disorder who lacked epileptic seizures. Developmental delay, intellectual disability, acquired microcephaly and structural brain abnormalities are common co-morbidities observed in this form of B6EEs [7, 67, 68, 137–139]. Systemic features like metabolic acidosis, anemia and gastrointestinal problems have been also described in PLPHP-deficient pateints [7, 67].

### *2.5.3 Biochemical features and diagnostic biomarkers*

Biochemical investigations performed in patient samples revealed amino acid and neurotransmitter abnormalities, reflecting the pleiotropic metabolic effects associated with altered PLP homeostasis. Among amino acids, elevated glycine in plasma and/or CSF was the most frequent alteration identified [7, 67, 68]. The enzyme that breaks down glycine, glycine cleavage system, requires PLP as a cofactor [140]. Abnormal monoamine neurotransmitter profile was detected in some patients, possibly due to suboptimal activity of the PLP-dependent enzyme AADC. Reported changes included low CSF levels of HVA (marker of low dopamine) and raised concentrations of 3-OMD, L-dopa, 5-HTP (CSF) and VLA (urine) indicating accumulation of AADC substrates [7, 67, 86]. Low PLP levels were detected in pretreatment plasma [68] and CSF [7] samples from two patients. Johnstone et al. [68] described accumulation of high levels of PNP in patient fibroblasts and PLPHPdeficient HEK293 cells. There is currently no established biomarker for this disease.
