Introduction
Hypophosphatasia (HPP) is a rare genetic disease caused by inactivating variants of the ALPL gene. Few data are available on the clinical presentation in Italy and/or on Italian HPP ...surveys.
Methods
There were 30 suspected HPP patients recruited from different Italian tertiary cares. Biological samples and related clinical, biochemical, and anamnestic data were collected and the ALPL gene sequenced. Search for large genomic deletions at the ALPL locus (1p36) was done. Phylogenetic conservation and modeling were applied to infer the effect of the variants on the protein structure.
Results
There were 21 ALPL variants and one large genomic deletion found in 20 out of 30 patients. Unexpectedly, NGS-driven differential diagnosis allowed uncovering three hidden additional HPP cases, for a total of 33 HPP subjects. Eight out of 24 coding variants were novel and classified as “pathogenic”, “likely pathogenic”, and “variants of uncertain significance”. Bioinformatic analysis confirmed that all the variants strongly destabilize the homodimer structure. There were 10 cases with low ALP and high VitB6 that resulted negative to genetic testing, whereas two positive cases have an unexpected normal ALP value. No association was evident with other biochemical/clinical parameters.
Discussion
We present the survey of HPP Italian patients with the highest ALPL mutation rate so far reported and confirm the complexity of a prompt recognition of the syndrome, mostly for HPP in adults. Low ALP and high VitB6 values are mandatory for the genetic screening, this latter remaining the gold standard not only to confirm the clinical diagnosis but also to make differential diagnosis, to identify carriers, to avoid likely dangerous therapy in unrecognized cases.
Inactivating mutations of CDC73 cause Hyperparathyroidism-Jaw Tumour syndrome (HPT-JT), Familial Isolated Hyperparathyroidism (FIHP) and sporadic parathyroid carcinoma. We conducted CDC73 mutation ...analysis in an HPT-JT family and confirm carrier status of the proband's daughter.
The proband had primary hyperparathyroidism (parathyroid carcinoma) and uterine leiomyomata. Her father and daughter had hyperparathyroidism (parathyroid adenoma) but no other manifestations of HPT-JT. CDC73 mutation analysis (sequencing of all 17 exons) and whole-genome copy number variation (CNV) analysis was done on leukocyte DNA of the three affecteds as well as the proband's unaffected sister.
A novel deletion of exons 4 to 10 of CDC73 was detected by CNV analysis in the three affecteds. A novel insertion in the 5'UTR (c.-4_-11insG) that co-segregated with the deletion was identified. By in vitro assay the 5'UTR insertion was shown to significantly impair the expression of the parafibromin protein. Screening for the mutated CDC73 confirmed carrier status in the proband's daughter and the biochemistry and ultrasonography led to pre-emptive surgery and resolution of the hyperparathyroidism.
A novel gross deletion mutation in CDC73 was identified in a three-generation HPT-JT family emphasizing the importance of including screening for large deletions in the molecular diagnostic protocol.
Brown tumors are osteolytic lesions associated with hyperparathyroidism (HPT). They may involve various skeletal segments, but rarely the cranio-facial bones. We report a case of a young boy with a ...swelling of the jaw secondary to a brown tumor presenting as the first manifestation of primary HPT (PHPT). He was found to have brown tumor located in the skull, as well. Different imaging technologies were employed for the diagnosis and follow-up after parathyroidectomy. We enclose a review of the literature on the employment of such imaging technologies in the differential diagnosis of osteolytic lesions. A multidisciplinary approach comprising clinical, laboratory and imaging findings is essential for the differential diagnosis of brown tumor in PHPT.
•Brown tumors are associated with more severe hyperparathyroidism (HPT).•The case of a young patient with primary HPT and brown tumors is reported.•A multidisciplinary approach should be employed for the differential diagnosis.•We reviewed the main radiological characteristics of different types of osteolytic lesions.
Context: Inactivating mutations of the calcium-sensing receptor (CASR) are implicated in different hypercalcemic syndromes, including familial hypocalciuric hypercalcemia (FHH), primary ...hyperparathyroidism (PHPT), and familial isolated hyperparathyroidism (FIHP). However, molecular diagnostics applied to large nonselected hypercalcemic cohorts from a single center have not been reported.
Objective: Our objective was to describe the prevalence, type, and potential pathogenicity of CASR mutations in a series of cases with FHH (n = 17), PHPT (n = 165), and FIHP (n = 3) and controls (n = 198) presenting at a single endocrine clinic.
Subjects: All were prospectively evaluated at the “Casa Sollievo della Sofferenza” Hospital in southern Italy over a 3-yr period.
Methods: CASR screening was conducted by denaturing HPLC. The variant CASRs were functionally characterized by transient transfection studies in kidney cells in vitro.
Results: A single novel missense variant was identified in one PHPT case. However, in FHH probands, mutations were found in eight of 17 (47%). With a hypercalcemic family member, mutation detection rate in FHH rose to seven of eight (87%), whereas only one of nine sporadic cases was positive, and none of the three FIHP cases had detectable CASR mutations. Five missense variant CASRs, identified in control subjects, performed as wild type in functional assays, whereas the missense mutant CASRs identified in the FHH patients, and in the one PHPT case, exhibited significant impairment. A novel intronic mutation (IVS4-19a→c) found in one FHH family, created an abnormally spliced product in an in vitro minigene assay.
Conclusion: CASR testing, with functional analysis, provides critical confirmatory evidence in the differential diagnosis of hypercalcemic states.
Prevalence, type, and potential pathogenicity of CASR mutations identified in a single endocrine clinic provide critical insights into the differential diagnosis of hypercalcemic states.
. Autoimmune polyglandular syndrome type 1 (APS-1) with or without reversible metaphyseal dysplasia is a rare genetic disorder due to inactivating variants of the autoimmune regulator,
gene. Clinical ...variability of APS-1 relates to pleiotropy, and the general dysfunction of self-tolerance to organ-specific antigens and autoimmune reactions towards peripheral tissues caused by the underlying molecular defect. Thus, early recognition of the syndrome is often delayed, mostly in cases with atypical presentation, and the molecular confirm through the genetic analysis of the
gene might be of great benefit.
. Our methods were to investigate, with a multigene panel next generation sequencing approach, two clinical cases, both presenting with idiopathic hypoparathyroidism, also comprising the
gene; as well as to comment our findings as part of a more extensive review of literature data.
. In the first clinical case, two compound heterozygote pathogenic variants of the
gene were identified, thus indicating an autosomal recessive inheritance of the disease. In the second case, only one
gene variant was found and an atypical dominant negative form of APS-1 suggested, later confirmed by further medical ascertainments.
. APS-1 might present with variable and sometimes monosymptomatic presentations and, if not recognized, might associate with severe complications. In this context, next generation diagnostics focused on a set of genes causative of partially overlapping disorders may allow early diagnosis.
Objective: To provide Italian expert opinion-based practical recommendations to improve the cooperation between clinicians and geneticists in order to optimize diagnostic flow and care of X-linked ...hypophosphatemia (XLH).
Methods: A panel of four geneticists and four clinicians from Italian reference centers for the diagnosis and management of XLH met virtually, first to highlight the critical issues in patient care and then to identify and share proposals to improve the diagnostic and care path of XLH.
Results: Critical issues emerged regarding the transfer of adequate clinical information from clinicians to geneticists, standardization and clarity of genetic reporting, and adequate interactions between clinicians and geneticists during patients’ follow-up. The necessary requirements for an appropriate request for evaluation of genetic variants and the need for a clear and clinically useful genetic report were agreed upon. Specifically designed template forms to be adopted with appropriate adjustments were defined and are here proposed for both the clinician’s request and the geneticist’s report.
Conclusions: The expert group strongly believes that collaboration between clinicians and geneticists should be encouraged in XLH, not only in the diagnostic phase but also during a patient’s follow-up, in order to manage patients more comprehensively and effectively.
Objective. Atypical parathyroid adenoma is a rare neoplasm, showing atypical histological features intermediate between classic benign adenoma and the rarest parathyroid carcinoma, whose the clinical ...behaviour and outcome is not yet understood or predictable. Up to date only two cases of atypical adenoma were found associated to a MEN1 syndrome, and only one was proved to carry a pathogenic variant of the MEN1 gene. Design. We report the clinical, histologic, and molecular findings of a 44-year-old woman, presenting with a histologically proved atypical parathyroid adenoma with an apparent aggressive behaviour. Methods and Results. CDC73 gene was screened at germline and somatic levels with no results. Whole exome sequencing performed on DNA extracted from blood leukocytes and tumour tissue revealed a somatic MEN1 gene heterozygous variant, c.912+1G > A, of the splicing donor site of exon 6. On immunohistochemistry, downregulation of the menin protein expression in the neoplastic cells was also observed. Conclusions. We report the second case of a rare association of a somatic MEN1 gene mutation in a patient with atypical parathyroid adenoma. We suggest that MEN1 gene could be an underestimate genetic determinant of these rare histological entities, and we highlight the utility of a complete genetic screening protocol, by the use of next-generation sequencing technology in such undetermined clinical cases with no frank clinical presentation.
Hereditary multiple exostosis represents the most frequent bone tumor disease in humans. It consists of cartilage deformities affecting the juxta-ephyseal region of long bones. Usually benign, ...exostosis could degenerate in malignant chondrosarcoma form in less than 5% of the cases. Being caused by mutations in the predicted tumor suppressor genes, EXT1 (chr 8q23-q24) and EXT2 (chr 11p11-p12) genes, HMEs are usually inherited with an autosomal dominant pattern, although “de novo” cases are not infrequent.
Here we present our genetic diagnostic report on the largest Southern Italy cohort of HME patients consisting of 90 subjects recruited over the last 5years.
Molecular screening performed by direct sequencing of both EXT1 and EXT2 genes, by MLPA and Array CGH analyses led to the identification of 66 mutations (56 different occurrences) and one large EXT2 deletion out of 90 patients (74.4%). The total of 21 mutations (20 different occurrences, 33.3%) and the EXT2 gene deletion were novel. In agreement with literature data, EXT1 gene mutations were scattered along all the protein sequence, while EXT2 lesions fell in the first part of the protein. Conservation, damaging prediction and 3-D modeling, in-silico, analyses, performed on three novel missense variants, confirmed that at least in two cases the novel aminoacidic changes could alter the structure stability causing a strong protein misfolding.
Here we present 20 novel EXT1/EXT2 mutations and one large EXT2 deletion identified in the largest Southern Italy cohort of patients affected by hereditary multiple exostosis.
► Molecular diagnostic for EXT1–EXT2 gene mutations was applied to 90 HME patients. ► Sixty-seven EXT1–EXT2 gene mutations were identified, in which twenty-one were novel. ► In-vitro assay functionally tested the pathogenicity of 3 EXT1 gene splicing variants.
Hyperparathyroidism Jaw-Tumour Syndrome (HPT-JT) is characterized by primary hyperparathyroidism (PHPT), maxillary/mandible ossifying fibromas and by parathyroid carcinoma in 15% of cases. ...Inactivating mutations of the tumour suppressor CDC73/HRPT2 gene have been found in HPT-JT patients and also as genetic determinants of sporadic parathyroid carcinoma/atypical adenomas and, rarely, typical adenomas, in familial PHPT. Here we report the genetic and molecular analysis of the CDC73/HRPT2 gene in three patients affected by PHPT due to atypical and typical parathyroid adenomas, in one case belonging to familial PHPT. Flag-tagged WT and mutant CDC73/HRPT2 proteins were transiently transfected in HEK293 cells and functional assays were performed in order to investigate the effect of the variants on the whole protein expression, nuclear localization and cell overgrowth induction. We identified four CDC73/HRPT2 gene mutations, three germline (c.679_680delAG, p.Val85_Val86del and p.Glu81_Pro84del), one somatic (p.Arg77Pro). In three cases the mutation was located within the Nucleolar Localisation Signals (NoLS). The three NoLS variants led to instability either of the corresponding mutated protein or mRNA or both. When transfected in HEK293 cells, NoLS mutated proteins mislocalized with a predeliction for cytoplasmic or nucleo-cytoplasmic localization and, finally, they resulted in overgrowth, consistent with a dominant negative interfering effect in the presence of the endogenous protein.
Single nucleotide polymorphisms (SNPs) of the calcium-sensing receptor (CASR) gene at the regulatory region were associated with idiopathic calcium nephrolithiasis. To confirm their association with ...nephrolithiasis, we tested patients with primary hyperparathyroidism (PHPT).
A genotype-phenotype association study.
In all, 332 PHPT patients and 453 healthy controls were genotyped for the rs7652589 (G>A) and rs1501899 (G>A) SNPs sited in the noncoding regulatory region of the CASR gene. Allele, haplotype, and diplotype distribution were compared between PHPT patients and controls, and in stone forming and stone-free PHPT patients.
The allele frequency at rs7652589 and rs1501899 SNPs was similar in PHPT patients and controls. The A minor alleles at these two SNPs were more frequent in stone forming (n=157) than in stone-free (n=175) PHPT patients (rs7652589: 36.9 vs 27.1%, P=0.007; rs1501899: 37.1 vs 26.4%, P=0.003). Accordingly, homozygous or heterozygous PHPT patients for the AA haplotype (n=174, AA/AA or AA/GG diplotype) had an increased stone risk (odds ratio 1.83, 95% confidence interval 1.2-2.9, P=0.008). Furthermore, these PHPT patients had higher serum concentrations of ionized calcium and parathyroid hormone (1.50 ± 0.015 mmol/l and 183 ± 12.2 pg/ml) than patients with the GG/GG diplotype (n=145, 1.47 ± 0.011 mmol/l (P=0.04) and 150 ± 11.4 pg/ml (P=0.049)). Using a logistic regression model, the increase in stone risk in PHPT patients was predicted by AA/AA or AA/GG diplotype, the highest tertile of serum ionized calcium values and the lowest tertile of age.
Polymorphisms located in the regulatory region of the CASR gene may increase susceptibility of the PHPT patients to kidney stone production.
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