Summary
We analyzed polymorphism of the
ALPL
gene in patients with low serum levels of tissue-nonspecific alkaline phosphatase (TNAP). The presence of three or more of the less frequent alleles of
...ALPL
polymorphisms was associated with significantly lower TNAP serum level and higher frequencies of metatarsal fractures, which may help confirm a clinical suspicion of adult hypophosphatasia.
Introduction
Alkaline phosphatases (ALPs) are membrane-bound enzymes that hydrolyze monophosphate esters at a high pH (pH 8–10). Inorganic pyrophosphate, pyridoxal 5-phosphate, the activated form of vitamin B
6
(PLP), and phosphoethanolamine (PEA), are natural substrates of ALPs. Hypophosphatasia (HPP, OMIM 146300, 241500, 241510) is a heterogeneous rare metabolic bone disease caused by loss-of-function mutations in the tissue-nonspecific alkaline phosphatase gene (
ALPL; MIM 171760
) with a deficiency of TNAP. Clinical presentation of HPP in adults demonstrated a wide range of manifestations, many of which are nonspecific. In the present study, we screened the polymorphic genetic variants of
ALPL
in 56 subjects presenting low serum levels of TNAP and/or other clinical signs of adult HPP in order to evaluate a possible role of polymorphic variants in the diagnosis and management of HPP in adults.
Methods
Genomic DNA was extracted from peripheral blood and
ALPL
gene was sequenced by PCR-based Sanger technique.
Results
Fourteen different polymorphic variants were found in the study population. A lower serum level of TNAP and higher frequencies of metatarsal fractures were observed in patients bearing three or more of the minor frequency alleles (MFAs) of the
ALPL
polymorphic variants. The presence of some MFAs, mostly as a contemporary presence of three or more of them, was found to be mainly represented in patients having both a significantly lower level of TNAP and a higher level of vitamin B6.
Conclusion
The genetic analysis and presence of some polymorphic variants may be an instrument to confirm clinical and biochemical data, consider adult HPP, and help clinicians be cautious in the administration of anti-reabsorption drugs.
Background
Normosmic congenital hypogonadotropic hypogonadism (ncHH) is caused by the deficient production, secretion, or action of gonadotropin‐releasing hormone (GnRH). Its typical clinical ...manifestation is delayed puberty and azoospermia. Homozygous and compound heterozygous mutations in the GNRHR gene (4q13.2) are the most frequent genetic causes of ncHH.
Objectives
(i) Characterization at the molecular level (genetic origin and functional effect) of a unique homozygous mutation (p.Gly99Glu) in a ncHH man; (ii) to provide a comprehensive catalog of GNRHR mutations with genotype–phenotype correlation and comparison of in vitro studies vs. in silico prediction tools.
Material and Methods
A ncHH man and his parents, in whom we performed the following: (i) Sanger sequencing, qPCR of the GNRHR gene; (ii) chromosome 4 SNP array; and (iii) competition binding assay and inositol phosphate signaling assay. PubMed and Human Genome Mutation Database (HGMD) search for GNRHR mutations. Bioinformatic analysis of 55 reported variants.
Results
qPCR showed two GNRHR copies in the index case. SNP array revealed the inheritance of two homologous chromosomes 4 from the mother (maternal heterodisomy; hUPD) with two loss of heterozygosity regions, one of them containing the mutated gene (maternal isodisomy; iUPD). Functional studies for the p.Gly99Glu mutation demonstrated a right‐shifted GnRH‐stimulated signaling response. Bioinformatic tools show that commonly used in silico tools are poor predictors of the function of ncHH‐associated GNRHR variants.
Discussion
Functional analysis of the p.Gly99Glu mutation is consistent with severely decreased GnRH binding affinity (a severe partial loss‐of‐function mutation). Complete LOF variants are associated with severe and severe/moderate phenotype, whereas partial LOF variants show wide range of clinical manifestations.
Conclusion
This is the first ncHH patient carrying a novel causative missense mutation of GNRHR with proven ‘severe pLOF’ due to maternal hUPD/iUPD of chromosome 4. Our literature review shows that functional studies remain essential both for diagnostic and potential therapeutic purposes.
Human disorders of phosphate (Pi) handling and skeletal mineralization represent a group of rare bone diseases. One of these disease is tumoral calcinosis (TC). In this study, we present the case of ...a patient with TC with a new
GALNT3
gene mutation. We also performed functional studies using an in vitro cellular model. Genomic DNA was extracted from peripheral blood collected from a teenage Caucasian girl affected by TC, and from her parents. A higher capability to form mineralization nodules in vitro was found in human preosteoblastic cells of mutant when compared to wild-type controls. We found a novel homozygous inactivating splice site mutation in intron I (c.516-2a>g). A higher capability to form mineralization nodules in vitro was found in the mutant cells in human preosteoblastic cells when compared to wild-type controls. Understanding the functional significance and molecular physiology of this novel mutation will help to define the role of FGF23 in the control of Pi homeostasis in normal and in pathological conditions.
Multiple endocrine neoplasia type 1 (MEN1) is a rare hereditary tumoral syndrome, featured by a combination of neoplasms of various endocrine and nonendocrine tissues. Approximately 33% of ...MEN1-related deaths are due to the malignant behaviour of well-differentiated neuroendocrine tumors (NETs), for which a preventive surgical treatment is not feasible. Somatostatin analogues (SSA) have been employed in the treatment of NETs in the stage of advanced or metastatic disease, in order to control the growth and secretion of tumor lesions. A longitudinal, open label study named "LARO-MEN1" was undertaken in order to assess whether early medical treatment with long-acting SSA could act as a preventive approach in small MEN1-related gastroenteropancreatic (GEP) NETs. Thirty consecutive patients affected by MEN1 were screened and 8 patients with small (<2 cm) NETs and abnormal laboratory values of at least one of the GEP hormones were administered octreotide acetate slow-release formulation (LAR) (10 mg i.m. every 28 days). Octreotide LAR was effective in decreasing GEP hormones and overall safe in the majority of patients up to six years of treatment, maintaining the disease stable also in terms of tumor size. The positive outcomes of this study in MEN1 patients reinforce the results obtained in advanced NETs on the use of SSA, opening to the opportunity for preventive use of octreotide LAR, aimed to delay or even avoid surgery in these patients.
Differently from other metabolic conditions, most of calcium metabolism disorders are diagnosed through simple detection of both serum and urinary excretion (24-h urine collection), levels of ...calcium, total and ionized form, and phosphate, and of calciotropic hormone serum levels, such as calcitonin, PTH and vitamin D metabolites. For the diagnosis and clinical monitoring of some metabolic bone diseases, such as osteoporosis and Paget's disease, the assessment of bone turnover is offering a useful tool for the evaluation of the therapeutic response in affected individuals. Markers of bone formation are represented by bone alkaline phosphatase and osteocalcin, while principal bone resorption markers are represented by pyridinoline, deoxypyridinoline and crosslinks of collagen N-telopeptide, both in the 24-h and fasting second morning urine collection. Only in selected conditions, here briefly reviewed, dynamic tests can offer an interpretation on the pathogenetic events causing a disorder of calcium metabolism.
PDB is genetically heterogeneous. Mutations of the sequestosome1 gene have been reported in sporadic and familial forms of Paget's in patients of French Canadian and British descent. Mutational ...analyses in different ethnic groups are needed to accurately investigate hereditary diseases. We describe two novel mutations of sequestosome1 in 62 Italian sporadic patients, confirming the role of the encoded protein in this disorder.
Introduction: Paget's disease of bone (PDB) is a relatively common disease of bone metabolism reported to affect up to 3% of whites over 55 years of age. The disorder is genetically heterogeneous, and at present, there is scientific evidence that at least eight different human chromosomal loci are correlated with its pathogenesis. Mutations of the sequestosome1 (SQSTM1) gene were identified as responsible for most of the sporadic and familial forms of Paget in patients of French Canadian and British descent. Such mutations were located at exon 7 and 8 levels, encoding for the ubiquitin protein‐binding domain (UBA) and representing a mutational hot spot area.
Materials and Methods: To verify the involvement of this gene in Italian subjects affected by PDB, we performed mutational analysis in 62 sporadic PDB cases.
Results: We described three different mutations at exon 8 level: P392L, already described in the French Canadian population and families predominantly of British descendent, and two novel mutations consisting of the amino acid substitutions M404V and G425R. No significant differences in the clinical history of PDB have been observed in patients with SQSTM1 mutations in respect to those without.
Conclusions: Even though our findings suggest a minor involvement of the SQSTM1 gene in the pathogenesis of sporadic Italian Paget's cases, the identification of different significant mutations within the SQSTM1 gene in unrelated, but clinically similar individuals, offers extremely convincing evidence for a causal relationship between this gene and PDB. Longitudinal studies are needed to assess the penetrance of genotype/phenotype correlations. Our findings confirm the evidence of a clustered mutation area at this level in this disorder.
Telomerase activity has been correlated to parathyroid carcinoma. Because its role in acquisition of a malignant phenotype by parathyroid cells is unclear, we treated telomerase‐positive cultured ...human parathyroid cancer cells with the telomerase inhibitor AZT, evaluating cell telomerase activity, cytotoxic effects, growth, and morphological changes. In vitro exposure of these cells to AZT correlated with inhibition of cell proliferation.
Introduction: Parathyroid carcinoma represents an uncommon cause of primary hyperparathyroidism, whose spectrum of clinical presentation, degree of malignancy, and prognosis are difficult to be properly identified. Neck surgery, specifically an en bloc resection of primary tumor, is the only curative treatment. Alternatively, affected patients could undergo repetitive palliative surgical exeresis of metastatic nodules. It has been previously shown that telomerase activity is specifically present in parathyroid carcinoma cells, being absent in hyperplastic and adenomatous tissues. Thus, determination of telomerase activity could represent either a useful diagnostic molecular marker for human parathyroid carcinoma or a potential target for pharmacological intervention in a malignant neoplasia usually resistant to chemo‐ and radiotherapeutic interventions.
Materials and Methods: To further investigate the role of telomerase activity in acquisition of a malignant phenotype by parathyroid cells, we treated telomeric repeat amplification protocol‐positive cultured human parathyroid cells with the telomerase inhibitor zidovudine, 3′‐azido‐3′deoxythymidine (AZT), evaluating cell telomerase activity, growth characteristics, potential cytotoxic effects, and morphological changes.
Results: Our findings indicate that in vitro exposure of human parathyroid cancer cells to AZT resulted in intracellular accumulation of AZT‐monophosphate (AZT‐MP) and inhibition of telomerase, which correlate with inhibition of human parathyroid cancer cell proliferation. Moreover, we also found that AZT induced an apoptotic rather than a necrotic type of cellular death. None of these effects were observed in human adenomatous parathyroid cells in culture.
Conclusions: Altogether these results indicate that AZT may be a highly effective agent against cancer parathyroid cells proliferation, which is an extremely important observation for a neoplasia which shows lack of response to classical pharmacological and physical antiblastic treatments.
The molecular mechanisms leading to adrenocortical tumorigenesis
have been only partially elucidated so far. Because the pituitary
hormone ACTH, via activation of the cAMP pathway, regulates both ...cell
proliferation/differentiation and steroid synthesis in the adrenal
cortex, in this study we focused on the cAMP-dependent transcription
factors cAMP responsive element modulator (CREM) and cAMP responsive
element binding protein (CREB). We studied CREM and CREB expression by
RT-PCR in human normal adrenal cortex (n = 3), adrenocortical
adenomas (n = 8), and carcinomas (n = 8). We found
transcripts corresponding to the isoforms α, β, γ, and τ2 of
the CREM gene in all of the normal adrenal tissues, in the adenomas,
and in seven of eight carcinomas. On the other hand, mRNA for the
inducible cAMP early repressor isoforms, which derive from an internal
promoter of CREM gene, was detected in the normal adrenal and in seven
of eight adenomas, but in only three of eight carcinomas. Similarly,
CREB transcripts were readily detectable in all normal adrenals and
adenomas, whereas they were not found in four of eight adrenal
carcinomas. To further characterize the carcinomas, telomerase activity
and the expression of the ACTH receptor gene were determined.
Telomerase activity in the carcinomas resulted in levels
significantly higher than in the adenomas, whereas the levels of ACTH
receptor mRNA were lower in the carcinomas. No correlation was found in
the carcinomas between the levels of the ACTH receptor transcript and
the loss of expression of CREB/inducible cAMP early repressor,
suggesting that this alteration is not secondary to an upstream
disregulation at the receptor level. In conclusion, our results suggest
that an alteration in cAMP signaling may be associated with
malignancies of the adrenal cortex.
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