Clinical and molecular description of the first Italian cohort of 33 subjects with hypophosphatasia

IntroductionHypophosphatasia (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.MethodsThere 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.ResultsThere 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.DiscussionWe 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

High Risk of Secondary Infections Following Thrombotic Complications in Patients With COVID-19

Background. This study’s primary aim was to evaluate the impact of thrombotic complications on the development of secondary infections. The secondary aim was to compare the etiology of secondary infections in patients with and without thrombotic complications. Methods. This was a cohort study (NCT04318366) of coronavirus disease 2019 (COVID-19) patients hospitalized at IRCCS San Raffaele Hospital between February 25 and June 30, 2020. Incidence rates (IRs) were calculated by univariable Poisson regression as the number of cases per 1000 person-days of follow-up (PDFU) with 95% confidence intervals. The cumulative incidence functions of secondary infections according to thrombotic complications were compared with Gray’s method accounting for competing risk of death. A multivariable Fine-Gray model was applied to assess factors associated with risk of secondary infections. Results. Overall, 109/904 patients had 176 secondary infections (IR, 10.0; 95% CI, 8.8–11.5; per 1000-PDFU). The IRs of secondary infections among patients with or without thrombotic complications were 15.0 (95% CI, 10.7–21.0) and 9.3 (95% CI, 7.9–11.0) per 1000-PDFU, respectively (P = .017). At multivariable analysis, thrombotic complications were associated with the development of secondary infections (subdistribution hazard ratio, 1.788; 95% CI, 1.018–3.140; P = .043). The etiology of secondary infections was similar in patients with and without thrombotic complications. Conclusions. In patients with COVID-19, thrombotic complications were associated with a high risk of secondary infections

Multi-integrated approach based on HPT-JT families for the identification of a set of biomarkers of Parathyroid Carcinoma

Introduzione. Nella sindrome da iperparatiroidismo associato a tumore della mandibola (HPT-JT), il carcinoma paratiroideo (PC) è causato da mutazioni del gene oncosoppressore CDC73 codificante per parafibromina, un componente del complesso PAF1 coinvolto nel rimodellamento della cromatina e nella regolazione del ciclo cellulare. Le mutazioni del gene MEN1 causano la sindrome omonima (MEN1), le cui lesioni paratiroidee sono benigne nel 99% dei casi ma, negli ultimi 40 anni, sono stati riportati 15 casi di associazione insolita fra PC e la sindrome MEN1 nel mondo. Il PC è un tumore raro e aggressivo per il quale le attuali terapie sono risultate inefficaci e l'asportazione chirurgica della lesione rimane l'unico approccio curativo. Tuttavia, la distinzione in prima diagnosi fra una lesione paratiroidea maligna e un’ iperplasia indolente o un’adenoma benigno rappresenta ancora oggi una sfida, in assenza di segni patognomici, come metastasi a distanza o recidive locali. Finora, sono stati compiuti sforzi infruttuosi per la ricerca di biomarcatorimolecolari che potessero indirizzare verso il (migliore) trattamento chirurgico (conservativo/demolitivo) al fine di ridurre il rischio di recidiva e di estendere la sopravvivenza libera da malattia. Scopo del progetto e metodi. Questo progetto mira a identificare le cause genetiche che portano allo sviluppo di un carcinoma paratiroideo (PC) e a individuare una serie di biomarcatori utili per una inequivocabile e precoce diagnosi di PC. A tal fine sono state applicate strategie di Next Generation Sequencing, quali il sequenziamento dell’ intero esoma e studi di espressione genica, a casi familiari (costituiti da uno o più soggetti affetti, portatori non affetti e controlli sani con lo stesso background genetico) piuttosto che a casi sporadici al fine di ridurre, per quanto possibile, la variabilità intrinseca causata dai diversi background genetici. La nostra rara coorte consta di 5 famiglie con HPT-JT e lesione paratiroidea maligna e mutazione costituzionale del gene CDC73 e una famiglia molto rara con mutazione MEN1 associata a PC. Risultati. Il sequenziamento dell’intero esoma ha mostrato che solo i soggetti affetti di 4 (delle 5) famiglie con HPT-JT reclutate per lo studio, condividono varianti rare (MAF <0,004) in geni codificanti per le integrine (ITGA3, ITGA2B, ITGA11, ITGAB6, ITGA9), recettori di superficie coinvolti nell'adesione cellulare alla matrice extracellulare (ECM) ed essenziali per la proliferazione, la sopravvivenza, l'adesione e la migrazione delle cellule. Inoltre sono state identificate ulteriori varianti nei geni che codificano per proteine coinvolte nel riparo del DNA come FANCC e BRIP1; NOTCH4; RET; BRCA1; BLK; MUC12; KMT2C; geni target della pathway di Hedgehog quali SMO, GLI3, mentre una variante del gene GLI2 è stata identificata solamente nei soggetti affetti della famiglia MEN1 associata a PC. Lo studio dell’ espressione genica è stato condotto confrontando i pazienti affetti vs controlli fra le famiglie con HPT-JT e l'unica famiglia MEN1-PC. L’ analisi effettuata ha mostrato un'espressione differenziale dei geni del sistema immunitario e inoltre ha evidenziato che i pazienti MEN1 e HPT-JT utilizzano diversi set di geni per controllare la mobilizzazione del calcio. Saggi funzionali. Cellule HEK293 sono state trasfettate con vettori di espressione codificanti la forma WT o mutata del gene CDC73: sono state testate 5 diverse mutazioni in presenza/assenza di Bortezomib, un farmaco già utilizzato in clinica per la terapia del mieloma multiplo. Questo inibitore del proteasoma sembra essere in grado di recuperare parzialmente l'espressione di proteine CDC73 mutate a causa di mutazioni missenso, in-frame e persino frameshift. Conclusioni e prospettive. Questo progetto, seppur preliminare, può aiutarci ad identificare biomarker utili per una diagnosi precoce e immediata di PC al fine di indirizzareverso il miglior approccio chirurgico e identificare i portatori asintomatici nelle famiglie affette per un intervento precoce ed efficace. Ipotizziamo che l'insorgenza/progressione/aggressività del PC possa essere dovuta alla deregolazione di proteine coinvolte nell'adesione cellula-cellula (come le integrine); allo squilibrio nell’ attivazione del sistema immunitario; alla deregolazione della pathway di Hedgehog e alla perdita di fattori scatenanti, come le proteine implicate nel mantenimento dell'integrità del DNA (FANCC, BRIP1, BRCA1). Infine, considerando che la ricerca di un farmaco efficace come approccio alternativo al trattamento chirurgico è risultata fino ad oggi vana, per la prima volta, riportiamo il possibile utilizzo di un farmaco chemioterapico ben conosciuto, il Bortezomib, per la terapia del PC dovuto a mutazioni del gene CDC73.Background. As part of the Hyperparathyroidism with Jaw Tumor syndrome (HPT-JT), parathyroid carcinoma (KP) is caused by mutations of the CDC73 tumor suppressor gene, encoding the parafibromin, a PAF1 co-transcription factor, involved in chromatin remodelling and cell cycle regulation. Mutations of MEN1 gene cause the namesake syndrome (MEN1), in whose parathyroid lesions are benign in 99% of cases: instead, in the last 40 ys, only 15 cases worldwide have been reported with the unusual association of KP in MEN1 syndrome. KP is a rare, aggressive life-threatening tumor for whose at the present current therapies resulted ineffective and the surgical removal of the lesion remains the only curative approach. However, to recognize in first diagnosis a malignant parathyroid lesion from an indolent hyperplasia or benign adenoma still represents a challenge, in absence of pathognomic signs, such as distant metastasis or local recurrences. So far, unsuccessful efforts have been made in search of clinical biomarkers that could address to the (best) surgery option (conservative/demolitive) in order to reduce the risk of recurrence and to extend the disease free survival. Purpose and methods. The present project aims to identify the genetic causes leading to the development of a parathyroid carcinoma (KP) and to detect a set of biomarkers for a possible unequivocal, early first diagnosis. We decided to apply high throughput strategies such Whole Exome Sequencing (WES) and Expression Profiling (EP) to familial cases (consisting of one or more affected subjects, non-affected carriers and healthy controls with the same genetic background) rather than to sporadic ones in order to, possibly, reduce the intrinsic variability caused by different genetic backgrounds. Our rare cohort consisted of 5 HPT-JT families with malignant parathyroid lesion and constitutional mutation of the CDC73 gene and a very rare family with MEN1 mutation associated with recurrent and familial KP. Results.WES analysis revealed that in 4 (out of 5) HPT-TJ families, surprisingly, only the affected subjects shared rare variants (MAF < 0.004) in genes encoding the integrins (ITGA3, ITGA2B, ITGA11, ITGAB6, ITGA9), cell surface receptors involved in cell adhesion to the extracellular matrix (ECM) and essential for proliferation, survival, adhesion and migration of cells. Moreover, other variants in genes encoding proteins involved in DNA repair such as FANCC and BRIP1; NOTCH4; RET; BRCA1; BLK; MUC12; KMT2C; Hedgehog target genes such as SMO, GLI3 were identified. Finally a variant in GLI2 gene was found inaffected subjects of MEN1- KP family. The EP analysis compared the affecteds vs controls between HPT-JT families and the unique MEN1-KP family. The analysis showed a differential expression of genes of immune system. The EP also evidenced that MEN1 and HPT-JT patients use different set of genes to control the calcium mobilization. Functional assay. Human embryonic kidney (HEK293) cell lines were transfected with eukaryotic expression vectors carrying WT or mutated CDC73 gene: 5 different mutations weretested in presence/absence of Bortezomib, a drug already used in clinic for the therapy of multiple mieloma. We proved that this proteasome inhibitor was able to partially rescue the expression of missense, inframe and even frameshift CDC73 gene deletions. Conclusions and perspectives. This preliminary project may help to find a biomarkers set for a prompt early diagnosis of KP, in order to suggest the best surgical approach and identify asymptomatic carriers in affected families for an efficient early intervention. We suppose that the onset/progression/aggressivity of KP may be due to the deregulation of proteins involved in cell-cell adhesion (such integrins); the derangement of immune system; the deregulation of the Hedgehog pathway and the lack of trigger factors, such as the proteins assigned to DNA integrity (FANCC, BRIP1, BRCA1). Finally we reported, for the first time, the possible use of a well known chemotherapy drug, the Bortezomib, for the therapy of KP CDC73 induced, taking into account that, so far, all the attempts to find an efficient drugs as alternative approach to the surgery, resulted ineffective

A Private 16q24.2q24.3 Microduplication in a Boy with Intellectual Disability, Speech Delay and Mild Dysmorphic Features

No data on interstitial microduplications of the 16q24.2q24.3 chromosome region are available in the medical literature and remain extraordinarily rare in public databases. Here, we describe a boy with a de novo 16q24.2q24.3 microduplication at the Single Nucleotide Polymorphism (SNP)-array analysis spanning ~2.2 Mb and encompassing 38 genes. The patient showed mild-to-moderate intellectual disability, speech delay and mild dysmorphic features. In DECIPHER, we found six individuals carrying a “pure” overlapping microduplication. Although available data are very limited, genomic and phenotype comparison of our and previously annotated patients suggested a potential clinical relevance for 16q24.2q24.3 microduplication with a variable and not (yet) recognizable phenotype predominantly affecting cognition. Comparing the cytogenomic data of available individuals allowed us to delineate the smallest region of overlap involving 14 genes. Accordingly, we propose ANKRD11, CDH15, and CTU2 as candidate genes for explaining the related neurodevelopmental manifestations shared by these patients. To the best of our knowledge, this is the first time that a clinical and molecular comparison among patients with overlapping 16q24.2q24.3 microduplication has been done. This study broadens our knowledge of the phenotypic consequences of 16q24.2q24.3 microduplication, providing supporting evidence of an emerging syndrome

Novel Pathogenic Variants of the AIRE Gene in Two Autoimmune Polyendocrine Syndrome Type I Cases with Atypical Presentation: Role of the NGS in Diagnostic Pathway and Review of the Literature

Background. 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, AIRE, 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 AIRE gene might be of great benefit. Methods. Our methods were to investigate, with a multigene panel next generation sequencing approach, two clinical cases, both presenting with idiopathic hypoparathyroidism, also comprising the AIRE gene; as well as to comment our findings as part of a more extensive review of literature data. Results. In the first clinical case, two compound heterozygote pathogenic variants of the AIRE gene were identified, thus indicating an autosomal recessive inheritance of the disease. In the second case, only one AIRE gene variant was found and an atypical dominant negative form of APS-1 suggested, later confirmed by further medical ascertainments. Conclusions. 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

Gonosomal Mosaicism for a Novel <i>COL5A1</i> Pathogenic Variant in Classic Ehlers-Danlos Syndrome

(1) Background: Classic Ehlers-Danlos syndrome (cEDS) is a heritable connective tissue disorder characterized by joint hypermobility and skin hyperextensibility with atrophic scarring. Many cEDS individuals carry variants in either the COL5A1 or COL5A2 genes. Mosaicism is relatively common in heritable connective tissue disorders but is rare in EDS. In cEDS, a single example of presumed gonosomal mosaicism for a COL5A1 variant has been published to date. (2) Methods: An 8-year-old girl with cEDS was analyzed by next-generation sequencing (NGS). Segregation was performed by Sanger sequencing in her unaffected parents. In the father, the mosaicism of the variant was further analyzed by targeted NGS and droplet digital PCR (ddPCR) in the blood and by Sanger sequencing in other tissues. (3) Results: The NGS analysis revealed the novel germline heterozygous COL5A1 c.1369G>T, p.(Glu457*) variant in the proband. Sanger chromatogram of the father’s blood specimen suggested the presence of a low-level mosaicism for the COL5A1 variant, which was confirmed by NGS and estimated to be 4.8% by ddPCR. The mosaicism was also confirmed by Sanger sequencing in the father’s saliva, hair bulbs and nails. (4) Conclusions: We described the second case of cEDS caused by paternal gonosomal mosaicism in COL5A1. Parental mosaicism could be an issue in cEDS and, therefore, considered for appropriate genetic counseling

Rare Somatic MEN1 Gene Pathogenic Variant in a Patient Affected by Atypical Parathyroid Adenoma

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

MEN1 gene mutation with parathyroid carcinoma: first report of a familial case

Background: The occurrence of parathyroid carcinoma in multiple endocrine neoplasia type I (MENI) is rare and the 15 cases of malignant parathyroid tumor reported so far have been associated with MENI in individuals and not with multiple members within a family. Methods: We report on a 61-year-old male, operated for a 7.3 cm parathyroid carcinoma infiltrating the esophagus. In his brother, a 4.6 cm parathyroid carcinoma was diagnosed histologically, while in the daughter, neck ultrasonography revealed 2 extrathyroidal nodules, yet to be excised. Results: Screening of the MEN1 gene identified a known germline heterozygous missense mutation (c.1252G>A; p.D418N) in exon 9, in all affected subjects. Conclusions: The occurrence of parathyroid carcinoma in more than one affected member of a single MEN1 family represents the first reported familial case. This suggests that additional constitutional genetic mutations may contribute to the variation in malignant potential and clinical behavior of parathyroid tumors in MEN1

Novel association of MEN1 gene mutations with parathyroid carcinoma

Inactivating mutations of the multiple endocrine neoplasia 1 (MEN1) gene cause MEN1 syndrome, characterized by primary hyperparathyroidism (pHPT), and parathyroid and gastro-entero-pancreatic pituitary tumors. At present, only 14 cases of malignant parathyroid tumor have been associated with the syndrome, with 6 cases carrying an inactivating mutation of the MEN1 gene. The present study presents the case of a 48-year-old female who presented with multigland pHPT and multiple pancreatic lesions. The patient underwent surgery several times for the excision of parathyroid hyperplasia, carcinoma and adenoma. The MEN1 gene was screened, revealing three variants (in cis) at the intron/exon 3 boundary (IVS2-3G>C, c.497A>T and c.499G>T) detected on the DNA of the proband, not shared by her relatives. RNA sequencing revealed that the IVS2-3C>G variant caused the skipping of the exon 3. Therefore, the present study reports on a novel rare association of MEN1 syndrome and parathyroid carcinoma. The reported splicing mutation was previously identified in subjects who always developed malignant lesions; thus, a possible genotype-phenotype association may be considered