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BACKGROUND: The application of massive parallel sequencing technologies in the molecular analysis of Charcot-Marie-Tooth (CMT) has enabled the rapid and cost-effective identification of numerous potentially significant variants for diagnostic purposes. The objective is to reduce the number of variants, focusing only on those with pathogenic significance. The 2015 American College of Medical Genetics and Genomics (ACMG) guidelines aid in achieving this goal, but it is now evident that a pathology or gene-specific review of these rules is essential to avoid misinterpretations that may result from blindly applying the criteria. This study demonstrates how revised ACMG criteria, combined with CMT-specific literature data and expertise, can alter the final classification of a variant. METHODS: We reviewed ACMG criteria based on current knowledge of CMT and provided suggestions for adapting them to the specificities of CMT. RESULTS: Of the 226 index patients analysed, a diagnostic yield of 20% was obtained. It is worth noting that the 9% of cases had their final diagnosis changed with the application of the revised criteria, often resulting in the loss of the pathogenic classification of a variant. CONCLUSIONS: The widespread availability of high-throughput sequencing technologies has enabled genetic testing even for laboratories without specific disease expertise. Disease-specific ACMG criteria can be a valuable tool to prevent the proliferation of variants of uncertain significance and the misinterpretation of variants.
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AIM: Biallelic mutations in the PTRH2 gene have been associated with infantile multisystem neurological, endocrine, and pancreatic disease (IMNEPD), a rare autosomal recessive disorder of variable expressivity characterized by global developmental delay, intellectual disability or borderline IQ level, sensorineural hearing loss, ataxia, and pancreatic insufficiency. Various additional features may be included, such as peripheral neuropathy, facial dysmorphism, hypothyroidism, hepatic fibrosis, postnatal microcephaly, cerebellar atrophy, and epilepsy. Here, we report the first Italian family presenting only predominant neurological features. METHODS: Extensive neurological and neurophysiological evaluations have been conducted on the two affected brothers and their healthy mother since 1996. The diagnosis of peripheral neuropathy of probable hereditary origin was confirmed through a sural nerve biopsy. Exome sequencing was performed after the analysis of major neuropathy-associated genes yielded negative results. RESULTS: Whole-exome sequencing analysis identified the homozygous substitution c.256C>T (p.Gln86Ter) in the PTRH2 gene in the two siblings. According to American College of Medical Genetics and Genomics (ACMG) guidelines, the variant has been classified as pathogenic. At 48 years old, the proband's reevaluation confirmed a demyelinating sensorimotor polyneuropathy with bilateral sensorineural hearing loss that had been noted since he was 13. Additionally, drug-resistant epileptic seizures occurred when he was 32 years old. No hepatic or endocrinological signs developed. The younger affected brother, 47 years old, has an overlapping clinical presentation without epilepsy. INTERPRETATION: Our findings expand the clinical phenotype and further demonstrate the clinical heterogeneity related to PTRH2 variants. We thereby hope to better define IMNEPD and facilitate the identification and diagnosis of this novel disease entity.
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Pancreatopatias , Humanos , Masculino , Itália , Feminino , Pancreatopatias/genética , Pancreatopatias/fisiopatologia , Pessoa de Meia-Idade , Códon sem Sentido , Linhagem , AdultoRESUMO
Background: Pheochromocytoma (Pheo) and paraganglioma (PGL) are rare tumors, mostly resulting from pathogenic variants of predisposing genes, with a genetic contribution that now stands at around 70%. Germline variants account for approximately 40%, while the remaining 30% is attributable to somatic variants. Objective: This study aimed to describe a new PHD2 (EGLN1) variant in a patient affected by metastatic Pheo and chronic myeloid leukemia (CML) without polycythemia and to emphasize the need to adopt a comprehensive next-generation sequencing (NGS) panel. Methods: Genetic analysis was carried out by NGS. This analysis was initially performed using a panel of genes known for tumor predisposition (EGLN1, EPAS1, FH, KIF1Bß, MAX, NF1, RET, SDHA, SDHAF2, SDHB, SDHC, SDHD, TMEM127, and VHL), followed initially by SNP-CGH array, to exclude the presence of the pathogenic Copy Number Variants (CNVs) and the loss of heterozygosity (LOH) and subsequently by whole exome sequencing (WES) comparative sequence analysis of the DNA extracted from tumor fragments and peripheral blood. Results: We found a novel germline PHD2 (EGLN1) gene variant, c.153G>A, p.W51*, in a patient affected by metastatic Pheo and chronic myeloid leukemia (CML) in the absence of polycythemia. Conclusions: According to the latest guidelines, it is mandatory to perform genetic analysis in all Pheo/PGL cases regardless of phenotype. In patients with metastatic disease and no evidence of polycythemia, we propose testing for PHD2 (EGLN1) gene variants. A possible correlation between PHD2 (EGLN1) pathogenic variants and CML clinical course should be considered.
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Neoplasias das Glândulas Suprarrenais , Leucemia Mielogênica Crônica BCR-ABL Positiva , Paraganglioma , Feocromocitoma , Policitemia , Neoplasias das Glândulas Suprarrenais/genética , Predisposição Genética para Doença , Mutação em Linhagem Germinativa/genética , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Paraganglioma/genética , Paraganglioma/patologia , Feocromocitoma/genética , Policitemia/genéticaRESUMO
Type 1 Chiari malformation (C1M) is characterized by cerebellar tonsillar herniation of 3-5 mm or more, the frequency of which is presumably much higher than one in 1000 births, as previously believed. Its etiology remains undefined, although a genetic basis is strongly supported by C1M presence in numerous genetic syndromes associated with different genes. Whole-exome sequencing (WES) in 51 between isolated and syndromic pediatric cases and their relatives was performed after confirmation of the defect by brain magnetic resonance image (MRI). Moreover, in all the cases showing an inherited candidate variant, brain MRI was performed in both parents and not only in the carrier one to investigate whether the defect segregated with the variant. More than half of the variants were Missense and belonged to the same chromatin-remodeling genes whose protein truncation variants are associated with severe neurodevelopmental syndromes. In the remaining cases, variants have been detected in genes with a role in cranial bone sutures, microcephaly, neural tube defects, and RASopathy. This study shows that the frequency of C1M is widely underestimated, in fact many of the variants, in particular those in the chromatin-remodeling genes, were inherited from a parent with C1M, either asymptomatic or with mild symptoms. In addition, C1M is a Mendelian trait, in most cases inherited as dominant. Finally, we demonstrate that modifications of the genes that regulate chromatin architecture can cause localized anatomical alterations, with symptoms of varying degrees.
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Malformação de Arnold-Chiari/genética , Montagem e Desmontagem da Cromatina/genética , Sequenciamento do Exoma , Mutação de Sentido Incorreto , Adolescente , Malformação de Arnold-Chiari/diagnóstico por imagem , Malformação de Arnold-Chiari/epidemiologia , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Sistema de Sinalização das MAP Quinases/genética , Imageamento por Ressonância Magnética , Masculino , Microcefalia/genética , Adulto JovemRESUMO
BACKGROUND: Charcot-Marie-Tooth disease (CMT) is a heterogeneous group of inherited peripheral neuropathies. Although the typical disease onset is reported in the second decade, earlier onsets are not uncommon. To date, few studies on pediatric populations have been conducted and the achievement of molecular diagnosis remains challenging. METHODS: During the last 24 years we recruited 223 patients with early-onset hereditary peripheral neuropathies (EOHPN), negative for PMP22 duplication, 72 of them referred by a specialized pediatric hospital. Genetic testing for CMT-associated genes has been carried out with a range of different techniques. RESULTS: Of the 223 EOHPN cases, 43% were classified as CMT1 (demyelinating), 49% as CMT2 (axonal), and 8% as CMTi (intermediate). Genetic diagnosis was reached in 51% of patients, but the diagnostic yield increased to 67% when focusing only on cases from the specialized pediatric neuromuscular centers. Excluding PMP22 rearrangements, no significant difference in diagnostic rate between demyelinating and axonal forms was identified. De novo mutations account for 38% of cases. CONCLUSIONS: This study describes an exhaustive picture of EOHPN in an Italian referral genetic center and analyzes the molecular diagnostic rate of a heterogeneous cohort compared with one referred by a specialized pediatric center. Our data identify MPZ, MFN2, GDAP1, and SH3TC2 genes as the most frequent players in EOHPN. Our study underlines the relevance of a specific neurological pediatric expertise to address the genetic testing and highlights its importance to clarify possible unexpected results when neuropathy is only a secondary clinical sign of a more complex phenotype.
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Doença de Charcot-Marie-Tooth , Humanos , Criança , Doença de Charcot-Marie-Tooth/diagnóstico , Doença de Charcot-Marie-Tooth/genética , Testes Genéticos , Fenótipo , MutaçãoRESUMO
The urban plan of Palermo (Sicily, Italy) has evolved throughout Punic, Roman, Byzantine, Arab, and Norman ages until it stabilized within the borders that correspond to the current historic center. During the 2012 to 2013 excavation campaign, new remains of the Arab settlement, directly implanted above the structures of the Roman age, were found. The materials investigated in this study derived from the so-called Survey No 3, which consists of a rock cavity of subcylindrical shape covered with calcarenite blocks: it was probably used to dispose of garbage during the Arabic age and its content, derived from daily activities, included grape seeds, scales and bones of fish, small animal bones, and charcoals. Radiocarbon dating confirmed the medieval origin of this site. The composition of the bacterial community was characterized through a culture-dependent and a culture-independent approach. Culturable bacteria were isolated under aerobic and anaerobic conditions and the total bacterial community was characterized through metagenomic sequencing. Bacterial isolates were tested for the production of compounds with antibiotic activity: a Streptomyces strain, whose genome was sequenced, was of particular interest because of its inhibitory activity, which was due to the Type I polyketide aureothin. Moreover, all strains were tested for the production of secreted proteases, with those belonging to the genus Nocardioides having the most active enzymes. Finally, protocols commonly used for ancient DNA studies were applied to evaluate the antiquity of isolated bacterial strains. Altogether these results show how paleomicrobiology might represent an innovative and unexplored source of novel biodiversity and new biotechnological tools. IMPORTANCE One of the goals of paleomicrobiology is the characterization of the microbial community present in archaeological sites. These analyses can usually provide valuable information about past events, such as occurrence of human and animal infectious diseases, ancient human activities, and environmental changes. However, in this work, investigations about the composition of the bacterial community of an ancient soil sample (harvested in Palermo, Italy) were carried out aiming to screen ancient culturable strains with biotechnological potential, such as the ability to produce bioactive molecules and secreted hydrolytic enzymes. Besides showing the biotechnological relevance of paleomicrobiology, this work reports a case of germination of putatively ancient bacterial spores recovered from soil rather than extreme environments. Moreover, in the case of spore-forming species, these results raise questions about the accuracy of techniques usually applied to estimate antiquity of DNA, as they could lead to its underestimation.
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Bactérias , Biodiversidade , Animais , Humanos , Sicília , Antibacterianos , Solo/químicaRESUMO
BACKGROUND: We report on a 20-week-old female fetus with a diaphragmatic hernia and other malformations, all of which appeared after the first-trimester ultrasound. METHODS AND RESULTS: Whole trio exome sequencing (WES) on cell-free fetal DNA (cff-DNA) revealed a de novo frameshift variant of the X-linked STAG2 gene. Loss-of-function (LoF) STAG2 variants cause either holoprosencephaly (HPE) or Mullegama-Klein-Martinez syndrome (MKMS), are de novo, and only affect females, indicating male lethality. In contrast, missense mutations associate with milder forms of MKMS and follow the classic X-linked recessive inheritance transmitted from healthy mothers to male offspring. STAG2 has been reported to escape X-inactivation, suggesting that disease onset in LoF females is dependent on inadequate dosing for at least some of the transcripts, as is the case with a part of the autosomal dominant diseases. Missense STAG2 variants produce a quantity of transcripts, which, while resulting in a different protein, leads to disease only in hemizygous males. Similar inheritance patterns are described for other escapee genes. CONCLUSIONS: This study confirms the advantage of WES on cff-DNA and emphasizes the role of the type of the variant in X-linked disorders.
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Achondrogenesis type II (ACG2) is a lethal skeletal dysplasia caused by dominant pathogenic variants in COL2A1. Most of the variants found in patients with ACG2 affect the glycine residue included in the Gly-X-Y tripeptide repeat that characterizes the type II collagen helix. In this study, we reported a case of a novel splicing variant of COL2A1 in a fetus with ACG2. An NGS analysis of fetal DNA revealed a heterozygous variant c.1267-2_1269del located in intron 20/exon 21. The variant occurred de novo since it was not detected in DNA from the blood samples of parents. We generated an appropriate minigene construct to study the effect of the variant detected. The minigene expression resulted in the synthesis of a COL2A1 messenger RNA lacking exon 21, which generated a predicted in-frame deleted protein. Usually, in-frame deletion variants of COL2A1 cause a phenotype such as Kniest dysplasia, which is milder than ACG2. Therefore, we propose that the size and position of an in-frame deletion in COL2A1 may be relevant in determining the phenotype of skeletal dysplasia.
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Acondroplasia/genética , Colágeno Tipo II/genética , Doenças Fetais/genética , Aborto Eugênico , Acondroplasia/diagnóstico , Acondroplasia/patologia , Acondroplasia/cirurgia , Adulto , Processamento Alternativo/genética , Feminino , Doenças Fetais/diagnóstico , Doenças Fetais/patologia , Doenças Fetais/cirurgia , Humanos , Imageamento Tridimensional , Itália , Mutação , Gravidez , Isoformas de Proteínas/genética , Deleção de Sequência , Ultrassonografia Pré-NatalRESUMO
BACKGROUND: Stickler Syndrome is a rare connective tissue disorder, characterized by clinical, and genetic heterogeneity. The clinical expression is highly variable, including moderate to severe myopia in childhood, hearing loss, facial dysmorphic features, cleft palate, and early osteoarthritis. COL2A1, COL11A1, and COL11A2 mutations account of the majority of autosomal dominant Stickler Syndrome and, in particular, a heterozygous mutation in COL11A1 gene is identified in about 10 to 20% of Stickler Syndrome patients. METHODS: Herein, we report a case of an 8-year- old child with Stickler Syndrome, presenting with early-onset of myopia with vitreal abnormalities, facial dysmorphic characteristics, and mild hearing loss later in childhood. To identify the underlying genetic cause, Whole Exome Sequencing was carried out for COL11A1 gene. RESULTS: A novel de novo heterozygous splice site variant (NM_001854: c.1845 + 5G> C) of the COL11A1 gene, which had not been previously reported, was identified by Whole Exome Sequencing. CONCLUSION: We reported a novel COL11A1 mutation in a child with Stickler Syndrome presenting a phenotype of early-onset of ocular anomalies and mild hearing loss later in childhood. Our findings confirm the variability of the expression of the disease, even in the contest of the same gene-related disorder, thus, contributing to improve the knowledge on clinical and molecular basis of this rare disease.
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Artrite/genética , Colágeno Tipo XI/genética , Doenças do Tecido Conjuntivo/genética , Perda Auditiva Neurossensorial/genética , Descolamento Retiniano/genética , Artrite/patologia , Criança , Doenças do Tecido Conjuntivo/patologia , Perda Auditiva Neurossensorial/patologia , Heterozigoto , Humanos , Masculino , Mutação , Fenótipo , Splicing de RNA , Descolamento Retiniano/patologiaRESUMO
This is the first case of NBAS disease detected by NBS for primary immunodeficiency. NBS with KRECs is revealing unknown potentialities detecting conditions that benefit from early recognition like NBAS deficiency. Immune phenotyping should be mandatory in patients with NBAS deficiency since they can exhibit severe immunodeficiency with hypogammaglobulinemia as the most frequent finding. Fever during infections is a known trigger of acute liver failure in this syndrome, so immune dysfunction, should never go unnoticed in NBAS deficiency in order to start adequate therapy and prophylaxis.