Severe hypophosphatasia: characterization of fifteen novel mutations in the ALPL gene

Hypophosphatasia is an inherited disorder characterized by defective bone mineralization and deficiency of serum and tissue liver/bone/kidney alkaline phosphatase (L/B/K ALP) activity. We report the characterization of ALPL gene mutations in a series of 11 families from various origins affected by perinatal and infantile hypophosphatasia. Sixteen distinct mutations were found, fifteen of them not previously reported: M45V, G46R, 388-391delGTAA, 389delT, T131I, G145S, D172E, 662delG, G203A, R255L, 876-881delAGGGGA, 962delG, E294K, E435K, and A451T. This confirms that severe hypophosphatasia is due to a large spectrum of mutations in Caucasian populations.     

低磷酸酶血症一家系组织非特异性碱性磷酸酶TNSALP基因突变分析

 目的 对1例儿童型低磷酸酶血症（HPP）患者及家系进行临床分析及基因突变检测,以探讨HPP的致病机制。方法 针对1例罕见的HPP患者的典型临床特点,进行实验室检验及影像学检查。进而收集患者及其亲属外周血,提取基因组DNA。针对ALPL基因12个外显子及附近内含子区合成引物,经PCR扩增后,直接对产物测序检测突变。结果 显示患者血碱性磷酸酶水平显著降低,骨骼具有佝偻病样改变;患者ALPL基因存在c.18delA及c.G407C两种突变。前者所致移码突变使得翻译提前终止,形成的截短蛋白 (p.V7Yfs18X)丧失了发挥酶活性及骨骼矿化作用的重要区域;而c.G407C导致其编码的氨基酸由精氨酸变为脯氨酸（R136P）。进一步检索PubMed及ALPL基因突变数据库,以上突变在国内外均未见报道。临床表现正常的患者母亲及祖母、父亲分别携带c.18delA和c.G407C突变,该家系符合常染色体隐性遗传。结论 ALPL基因c.18delA和c.G407C两种新突变,与HPP临床表现密切相关。     

Utility of genetic testing for prenatal presentations of hypophosphatasia

Hypophosphatasia (HPP) is a rare inherited disease affecting bone and dental mineralization due to loss-of-function mutations in the ALPL gene encoding the tissue nonspecific alkaline phosphatase (TNSALP). Prenatal benign HPP (PB HPP) is a rare form of HPP characterized by in utero skeletal manifestations that progressively improve during pregnancy but often still leave symptoms after birth. Because the prenatal context limits the diagnostic tools, the main difficulty for clinicians is to distinguish PB HPP from perinatal lethal HPP, the most severe form of HPP.We previously attempted to improve genotype phenotype correlation with the help of a new classification of variants based on functional testing. Among 46 perinatal cases detected in utero or in the neonatal period for whose ALPL variants could be classified, imaging alone was thought to clearly diagnose severe lethal HPP in 35 cases, while in 11 cases, imaging abnormalities could not distinguish between perinatal lethal and BP HPP. We show here that our classification of ALPL variants may improve the ability to distinguish between perinatal lethal and PB HPP in utero.     

Infantile hypophosphatasia without bone deformities presenting with severe pyridoxine-resistant seizures

An infant carrying a heterozygous c.43_46delACTA and a heterozygous c.668 G>A mutation in the ALPL gene with hypophosphatasia in the absence of bone deformities presented with therapy-resistant seizures. Pyridoxal phosphate was extremely high in CSF and plasma. Pyridoxine treatment had only a transient effect and the severe encephalopathy was fatal. Repeated brain MRIs showed progressive cerebral damage. The precise metabolic cause of the seizures remains unknown and pyridoxine treatment apparently does not cure the epilepsy.     

Perinatal hypophosphatasia: radiology, pathology and molecular biology studies in a family harboring a splicing mutation (648+1A) and a novel missense mutation (N400S) in the tissue-nonspecific alkaline phosphatase (TNSALP) gene.

We report on a postmortem diagnosis of perinatal lethal hypophosphatasia, an inborn error of metabolism characterized by a liver/bone/kidney alkaline phosphatase (ALP)-related defective bone mineralization due to mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene. Radiological and pathological studies identified a perinatal lethal hypophosphatasia showing a generalized bone mineralization defect including asymmetry of the cervical vertebral arches in a 22 +4 weeks' gestation fetus. Both parents revealed low serum ALP activities supporting the diagnosis. Sequencing analysis of the TNSALP gene showed two heterozygous mutations, 648+1A, a mutation affecting the donor splice site in exon 6, and N400S, a novel missense mutation in exon 11, located near the active site and very close to histidins 364 and 437, two crucial residues of the active site. Sequencing of exons 6 and 11 in the parents showed that 648+1A was from maternal origin and N400S from paternal origin. DNA-based prenatal testing in the subsequent pregnancy following a chorionic villous sampling performed at 10 weeks of gestation showed no mutation and a healthy infant was born at term.     

Perinatal hypophosphatasia: Radiology,pathology and molecular biology studies in a family harboring a splicing mutation (648+1A) and a novel missense mutation (N400S) in the tissue‐nonspecific alkaline phosphatase (TNSALP) gene

We report on a postmortem diagnosis of perinatal lethal hypophosphatasia, an inborn error of metabolism characterized by a liver/bone/kidney alkaline phosphatase (ALP)‐related defective bone mineralization due to mutations in the tissue‐nonspecific alkaline phosphatase (TNSALP) gene. Radiological and pathological studies identified a perinatal lethal hypophosphatasia showing a generalized bone mineralization defect including asymmetry of the cervical vertebral arches in a 22 +4 weeks' gestation fetus. Both parents revealed low serum ALP activities supporting the diagnosis. Sequencing analysis of the TNSALP gene showed two heterozygous mutations, 648+1A, a mutation affecting the donor splice site in exon 6, and N400S, a novel missense mutation in exon 11, located near the active site and very close to histidins 364 and 437, two crucial residues of the active site. Sequencing of exons 6 and 11 in the parents showed that 648+1A was from maternal origin and N400S from paternal origin. DNA‐based prenatal testing in the subsequent pregnancy following a chorionic villous sampling performed at 10 weeks of gestation showed no mutation and a healthy infant was born at term. © 2001 Wiley‐Liss, Inc.     

Prevalence of c.1559delT in ALPL, a common mutation resulting in the perinatal (lethal) form of hypophosphatasia in Japanese and effects of the mutation on heterozygous carriers

Hypophosphatasia (HPP) is an inherited disorder caused by mutations in ALPL that encodes an isozyme of alkaline phosphatase (ALP), TNSALP. One of the most frequent ALPL mutations is c.1559delT, which causes the most severe HPP, the perinatal (lethal) form (pl-HPP). c.1559delT has been found only in Japanese and its prevalence is suspected to be high; however, the allele frequency of c.1559delT in Japanese remains unknown. We designed a screening system for the mutation based on high-resolution melting curve analysis, and examined the frequency of c.1559delT. We found that the c.1559delT carrier frequency is 1/480 (95% confidence interval, 1/1562-1/284). This indicates that ～1 in 900?000 individuals to have pl-HPP caused by a homozygous c.1559delT mutation. In our analysis, the majority of c.1559delT carriers had normal values of HPP biochemical markers, such as serum ALP and urine phosphoethanolamine. Our results indicate that the only way to reliably detect whether individuals are pl-HPP carriers is to perform the ALPL mutation analysis.     

A novel missense mutation of the tissue-nonspecific alkaline phosphatase gene detected in a patient with hypophosphatasia

Hypophosphatasia is a rare heritable inborn error of metabolism characterized by abnormal bone mineralization associated with a deficiency of alkaline phosphatase. The clinical expression of hypophosphatasia is highly variable, ranging from death in utero to pathologic fractures first presenting in adulthood. We investigated the tissue-nonspecific alkaline phosphatase (TNSALP) gene from a Japanese female patient with hypophosphatasia. By a quantitative polymerase chain reaction (PCR) method, the amount of TNSALP mRNA appeared to be almost equal to that in normal individuals. Gene analysis clarified that the hypophosphatasia originated from a missense mutation and a nucleotide deletion. The missense mutation, a C ? T transition at position 1041 of cDNA, results in an amino acid change from Leu to Phe at codon 272, which has not yet been reported. The previously reported deletion of T at 1735 causes a frame shift mutation downstream from Leu at codon 503. Family analysis showed that the mutation 1041T and the deletion 1735T had been inherited from the proband's father and mother, respectively. An expression experiment revealed that the mutation 1041T halved the expression of alkaline phosphatase activity. Using homology analysis, the Leu-272 was confirmed to be highly conserved in other mammals.     

A novel mutation in the dihydrolipoamide dehydrogenase E3 subunit gene (DLD) resulting in an atypical form of alpha-ketoglutarate dehydrogenase deficiency

The alpha-ketoglutarate dehydrogenase complex (KGDC) catalyses the decarboxylation of alpha-ketoglutarate into succinyl-coenzyme A in the Krebs cycle. This enzymatic complex is made up of three subunits (E1, encoded by PDHA1; E2, encoded by DLST; and E3, encoded by DLD). The E3 subunit is common to two other enzymatic complexes, namely pyruvate dehydrogenase complex (PDC) and branched-chain ketoacid dehydrogenase complex (BCKDC). KGDC deficiency is a rare autosomal recessive disorder, most often presenting with severe encephalopathy and hyperlactatemia with neonatal onset. We found a KGDC deficiency in cultured skin fibroblasts from three siblings born to consanguinous parents. E3 subunit activity was shown to be deficient (20% of control values), despite the absence of usual clinical clues to E3 deficiency, i.e. accumulation of pyruvate and branched-chain amino acids in plasma and branched-chain alpha-ketoacids in urine. RT-PCR of E3 mRNA from the three patients, followed by sequencing, revealed an homozygous c.1444A>G substitution located in E3 exon 13, predictive of a p.R482G (or R447G in the processed gene product) substitution in a highly conserved domain of the protein. Only eleven E3 mutations have been reported so far. The only other case of E3 deficiency without clinical or biochemical evidences of PDC and BCKDC deficiencies has been ascribed to a c.1436A>T (p.D479V; or D444V in the processed gene product) mutation, very close to the mutation reported herein. Since c.1444A>G (p.R482G; or R447G in the processed gene product) and c.1436A>T (p.D479V; or D444V in the processed gene product) lie within the interface domain of E3 with E2 (KGDC and BCKDC) or the E3-binding protein (PDC), our data suggest that interaction of E3 with these other subunits differs in some extent among KGDC, PDC, and BCKDC.     

Phenotypically dissimilar hypophosphatasia in two sibships

Autosomal dominant and autosomal recessive forms of hypophosphatasia have been reported; generally the clinical picture runs true to form in families. In each of 2 kindreds, 2 sibs were clinically affected by hypophosphatasia to a markedly different extent. One set of sibs showed the lethal (perinatal) and infantile forms. The other showed the dental and adult forms. In both families there was consanguinity, albeit distant, and clinical expression in sibs supporting autosomal recessive inheritance.     

Phenotypically dissimilar hypophosphatasia in two sibships.

Autosomal dominant and autosomal recessive forms of hypophosphatasia have been reported; generally the clinical picture runs true to form in families. In each of 2 kindreds, 2 sibs were clinically affected by hypophosphatasia to a markedly different extent. One set of sibs showed the lethal (perinatal) and infantile forms. The other showed the dental and adult forms. In both families there was consanguinity, albeit distant, and clinical expression in sibs supporting autosomal recessive inheritance.     

Denaturing gradient gel electrophoresis analysis of the tissue nonspecific alkaline phosphatase isoenzyme gene in hypophosphatasia

Hypophosphatasia, a heritable form of rickets/osteomalacia, was first described in 1948. The biochemical hallmark, subnormal alkaline phosphatase (ALP) activity in serum, reflects a generalized disturbance involving the tissue-nonspecific isoenzyme of ALP (TNSALP). Deactivating mutations in the gene that encodes TNSALP have been reported in patients worldwide. Nevertheless, hypophosphatasia manifests an extraordinary range of clinical severity spanning death in utero to merely premature loss of adult teeth. There is no known medical treatment. To delineate the molecular pathology which explains the disease variability and to clarify the pattern(s) of inheritance for mild cases of hypophosphatasia, we developed comprehensive mutational analysis of TNSALP. High efficiency of mutation detection was possible by denaturing gradient gel electrophoresis (DGGE). Primers and conditions were established for all TNSALP coding exons (2-12) and adjacent splice sites so that the amplicons incorporated a GC clamp on one end. For each amplicon, the optimum percentage denaturant was determined by perpendicular DGGE. In 19 severely affected pediatric subjects (having perinatal or infantile hypophosphatasia or early presentation during childhood) from among our large patient population, we detected 2 TNSALP mutations each in 16 patients (84%) as expected for autosomal recessive disease. For 2 patients (11%), only 1 TNSALP mutation was detected by DGGE. However, one subject (who died from perinatal hypophosphatasia) had a large deletion as the second mutation. In the other (with infantile hypophosphatasia), no additional mutation was detected by DNA sequencing of all protein-coding exons. Possibly, she too has a deletion. For the final patient, with unclassifiable hypophosphatasia (5%), we detected only a single mutation which has been reported to cause relatively mild autosomal dominant disease; the other allele appeared to be intact. Hence, DGGE analysis was 100% efficient in detecting mutations in the coding exons and adjacent splice sites of TNSALP in this group of severely affected patients but, as expected, failed to detect a large deletion. To date, at least 78 different TNSALP mutations (in about 70 hypophosphatasia patients) have been reported globally. In our large subset of severely affected patients, we identified 8 novel TNSALP mutations (Ala34Ser, Val111Met, Delta G392, Thr117His, Arg206Gln, Gly322Arg, Leu397Met, and Gly409Asp) and 1 new TNSALP polymorphism (Arg135His) furthering the considerable genotypic variability of hypophosphatasia.     

Specific ultrasonographic features of perinatal lethal hypophosphatasia

Prenatal diagnosis of perinatal lethal hypophosphatasia (PL-HPH) by ultrasonography is difficult as PL-HPH must be differentiated from other skeletal dysplasias with short long bones and poor mineralization of the skeleton, such as osteogenesis imperfecta type II and achondrogenesis/hypochondrogenesis. Here we present a case of molecularly confirmed PL-HPH and illustrate specific ultrasonographic findings that help to distinguish PL-HPH from similar conditions.     

First identification of a gene defect for hypophosphatasia: evidence that alkaline phosphatase acts in skeletal mineralization

Hypophosphatasia is a heritable disorder characterized by defective osteogenesis and deficient liver/bone/kidney alkaline phosphatase (L/B/K ALP) activity. Severe forms of the disease are inherited in an autosomal recessive fashion. We examined cultured skin fibroblasts from twelve patients with severe hypophosphatasia. All were deficient in L/B/K ALP activity, yet produced normal levels of the corresponding mRNA. Sequence analysis of L/B/K ALP cDNA isolated from one of the patient-derived fibroblast lines revealed a point mutation that converted amino acid 162 of mature L/B/K ALP from alanine to threonine. The patient was homozygous and the parents, who are second cousins, heterozygous for this mutation. Introduction of the mutation into an otherwise normal cDNA disrupted the expression of active enzyme, demonstrating that a defect in the L/B/K ALP gene resulted in hypophosphatasia and that the enzyme is, therefore, essential for normal skeletal mineralization.     

Discordant fetal phenotype of hypophosphatasia in two siblings

Hypophosphatasia (HPP) is an autosomal recessive metabolic disorder with impaired bone mineralization due to mutations in the ALPL gene. The genotype‐phenotype correlation of this disorder has been widely described. Here, we present two affected siblings, whose fetal phenotypes were discordant. A 31‐year‐old Japanese woman, G0P0, was referred to our institution because of fetal micromelia. After obstetric counseling, the pregnancy was terminated at 21 weeks’ gestation. Post‐mortem radiographs demonstrated severely defective mineralization of the skeleton. The calvarial, spinal, and tubular bones were mostly missing. Only the occipital bones, mandible, clavicles, ribs, one thoracic vertebra, ilia, and tibia were relatively well ossified. The radiological findings suggested lethal HPP. Genetic testing for genomic DNA extracted from the umbilical cord identified compound heterozygous mutations in the ALPL gene (c.532T>C, p.Y178H; c.1559delT, p.Leu520Argfs*86). c.532T>C was a novel variant showing no residual activity of the protein by the functional analysis. The parents were heterozygous carriers. In the next pregnancy, biometric values on fetal ultrasonography at 20 and 26 weeks’ gestation were normal. At 34 weeks, however, a small chest and shortening of distal long bones came to attention. The neonate delivered at 41 weeks showed serum ALP of <5U/L. Radiological examination showed only mild thoracic hypoplasia and metaphyseal mineralization defects of the long bones. ALP replacement therapy was introduced shortly after birth, and the neonate was discharged at day 22 without respiratory distress. Awareness of discordant fetal phenotypes in siblings with HPP precludes a diagnostic error, and enables early medical intervention to mildly affected neonates.     

Matrix vesicles in osteomalacic hypophosphatasia bone contain apatite-like mineral crystals.

Hypophosphatasia, a heritable disease characterized by deficient activity of the tissue nonspecific isoenzyme of alkaline phosphatase (TNSALP), results in rickets and osteomalacia. Although identification of TNSALP gene defects in hypophosphatasia establishes a role of ALP in skeletal mineralization, the precise function remains unclear. The initial site of mineralization (primary mineralization) normally occurs within the lumen of TNSALP-rich matrix vesicles (MVs) of growth cartilage, bone, and dentin. We investigated whether defective calcification in hypophosphatasia is due to a paucity and/or a functional failure of MVs secondary to TNSALP deficiency. Nondecalcified autopsy bone and growth plate cartilage from five patients with perinatal (lethal) hypophosphatasia were studied by nondecalcified light and electron microscopy to assess MV numbers, size, shape, and ultrastructure and whether hypophosphatasia MVs contain apatite-like mineral, as would be the case if these MVs retained their ability to concentrate calcium and phosphate internally despite a paucity of TNSALP in their investing membranes. We found that hypophosphatasia MVs are present in approximately normal numbers and distribution and that they are capable of initiating internal mineralization. There is retarded extravesicular crystal propagation. Thus, in hypophosphatasia the failure of bones to calcify appears to involve a block of the vectorial spread of mineral from initial nuclei within MVs, outwards, into the matrix. We conclude that hypophosphatasia MVs can concentrate calcium and phosphate internally despite a deficiency of TNSALP activity.     

Diagnosis of two neonates with galactosemia by using next generation sequencingCSCD

Objective To explore the genetic basis of two neonates suspected for galactosemia. Methods Next generation sequencing (NGS) was used to screen the whole exome of the neonates. Suspected mutation was validated by PCR and Sanger sequencing. Potential impact of novel mutation was predicted by using PolyPhen-2, MutationTaste and SIFT software. Results Both neonates harbored compound heterozygous mutations of the GALT gene inherited from their parents. One has inherited two novel mutations c. 564G>C(p. Q188H) and c. 116A>T(p. D39V) respectively from his father and mother. The other has inherited mutations c. 754C>T(p. Q252X) and c. 904 + 1OT from her father and mother, respectively. Conclusion The galactosemia in the two neonates may be attributed to compound heterozygous mutations of the GALT gene. This is the first domestic report of using the NGS for the diagnosis of galactosemia. © 2018 West China University of Medical Sciences. All rights reserved.     

Identification of novel mutations in the PCCB gene in European propionic acidemia patients

Propionyl‐CoA carboxylase (PCC) is a biotin‐dependent enzyme located in the mitochondrial matrix. Mutations in the PCCA and PCCB genes, which encode the a and b subunits of this heteropolymer, result in propionic acidemia (PA). We report the molecular analysis of b‐deficient patients from Spain and Austria. Subjects were screened for defects affecting the PCCB gene by direct sequencing from genomic PCR products, restriction digests and mRNA analysis by RT‐PCR. Study by western blot of the presence of immunoreactive b‐PCC protein was also performed. A total of four novel sequence variations were found including the point mutations V205D, and M442T, and the frameshift mutation 790‐791insG. Additionaly, a new point change, L17M, was identified on the same allele as 790‐791insG. The missense changes described above were not found in at least 40 control chromosomes analyzed. The Austrian patients were homozygous for V205D. One of the Spanish subjects was heterozygous for M442T and the known mutation c1170insT. The other Spanish patient carried L17M+790‐791insG on one allele, and the described mutation E168K on the other mutant chromosome. The mutations V205D and M442T were confirmed at RNA level and also we have detected the presence of immunoreactive b‐PCC protein translated from these mutant alleles. The patient having L17M+790‐791insG and E168K also presented immunoreactive b‐PCC protein. However, no cDNA product was obtained from the chromosome carrying L17M+790‐791insG. We propose that 790‐791insG, which causes a frameshift and a premature stop codon, is responsible for this finding. In any case, the translation from this mutant cDNA would produce a severily truncated peptide and, in consequence, a non‐functional protein. Expression analysis of all these changes will help us to clarify their structural/functional consequences. Hum Mutat 14:89–90, 1999. © 1999 Wiley‐Liss, Inc.     

Perinatal hypophosphatasia: diagnosis and detection of heterozygote carriers within the family

We report on two families in which one or two children had a severe disorder of skeletal development detected by prenatal ultrasonography. The children died postnatally and showed typical radiological and biochemical findings of perinatal hypophosphatasia. Biochemical analysis revealed a low activity of alkaline phosphatase (AP) and a high value of pyridoxal-5-phosphate (PLP), one of its natural substrates. The screening for mutations of the tissue nonspecific alkaline phosphatase (TNSALP) gene showed homozygosity for a point mutation (G 317 --> D) in the two affected children of the first family. The affected child of the second family was homozygous for a nonsense mutation (R 411 --> X). Family screening revealed that the determination of AP and PLP is helpful for detection of heterozygotes. However, heterozygote children had values of AP in the lower normal range during phases of rapid growth. The determination of PLP proved to be more sensitive in these cases. It should be kept in mind that during the last trimester of gestation there is an increase in maternal AP activity and a normalization of PLP due to placental AP, which is not affected. Therefore, in the course of a prenatal diagnosis in an index case, paternal blood should be analyzed in parallel. For detailed genetic counseling and early prenatal diagnosis in following pregnancies, the possibility of mutation analysis should be used.     

A molecular-based estimation of the prevalence of hypophosphatasia in the European population

The prevalence of hypophosphatasia (HP), a rare metabolic disorder due to loss-of-function mutations in the ALPL gene, has never been estimated in the European population. Only one published study evaluated the incidence of severe HP at 1/100,000 in Canada 53 years ago. Moderate forms of hypophosphatasia (mHP), including HP with moderate bone features and the mildest form odontohypophosphatasia, reflect both recessive and dominant inheritance, and are therefore expected to be more frequent than severe forms of HP. Here we estimated both the prevalences of severe and mHP in European populations. The prevalence of severe HP was estimated at 1/300,000 on the basis of the number of cases tested in our laboratory and originating from France during the period 2000-2009. The prevalence of mHP was then estimated by using the proportion of dominant mutations among severe alleles and by estimating the penetrance of the disease in heterozygotes for dominant mutations. According to a genetic model with four alleles resulting in 10 distinct genotypes, the prevalence of dominant mHP in the European population was estimated to be 1/6370, pointing out that mHP is much more frequent than severe HP.