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Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive genetic defects in cortisol synthesis and shows elevated ACTH concentrations, which in turn has downstream effects. The most common variant of CAH, 21-hydroxylase deficiency (21OHD), is the result of pathogenic variants in the CYP21A2 gene and is one of the most common monogenic disorders. However, the genetics of 21OHD is complex and challenging. The CYP21A2 gene is located in the RCCX copy number variation (CNV), a complex, multiallelic, and tandem CNV in the major histocompatibility complex (MHC) class III region on chromosome 6 (band 6p21.3). Here, CYP21A2 and its pseudogene CYP21A1P are located 30 kb apart and share a high nucleotide homology of approximately 98% and 96% in exons and introns, respectively. This high-sequence homology facilitates large structural rearrangements, copy number changes, and gene conversion through intergenic recombination. There is a good genotype-phenotype correlation in 21OHD, and genotyping can be performed to confirm the clinical diagnosis, predict long-term outcomes, and determine genetic counseling. Thus, genotyping in CAH is clinically relevant but the interpretations can be challenging for non-initiated clinicians. Here, there are some concrete examples of how molecular diagnosis can sometimes require the use of multiple molecular strategies.
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We describe the complex case of a 44-year-old man with polycystic kidney disease, mild cognitive impairment, and tremors in the upper limbs. Brain MRI showed lesions compatible with leukodystrophy. The diagnostic process, which included clinical exome sequencing (CES) and chromosomal microarray analysis (CMA), revealed a triple diagnosis: autosomal dominant polycystic kidney disease (ADPKD) due to a pathogenic variant, c.2152C>T-p.(Gln718Ter), in the PKD1 gene; late-onset phenylketonuria due to the presence of two missense variants, c.842C>T-p.(Pro281Leu) and c.143T>C-p.(Leu48Ser) in the PAH gene; and a 915 Kb duplication on chromosome 15. Few patients with multiple concurrent genetic diagnoses are reported in the literature; in this ADPKD patient, genome-wide analysis allowed for the diagnosis of adult-onset phenylketonuria (which would have otherwise gone unnoticed) and a 15q11.2 duplication responsible for cognitive and behavioral impairment with incomplete penetrance. This case underlines the importance of clinical genetics for interpreting complex results obtained by genome-wide techniques, and for diagnosing concurrent late-onset monogenic conditions.
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Disfunção Cognitiva , Doenças Desmielinizantes , Deficiência Intelectual , Transtornos do Metabolismo dos Lipídeos , Doenças por Armazenamento dos Lisossomos , Doenças Neurodegenerativas , Fenilcetonúrias , Rim Policístico Autossômico Dominante , Adulto , Masculino , Humanos , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/genética , Cromossomos Humanos Par 15 , Transtornos de Início TardioRESUMO
Next generation sequencing (NGS) is a widespread molecular biology method integrated into clinical practice to detect genetic variants, for diagnostic and prognostic purposes. The scheduled external quality assessments (EQA) is integral part of clinical molecular laboratory quality assurance. The EQA provides an efficient system to compare analytic test performances among different laboratories, which is essential to evaluate consistency of molecular test. EQA failures demands targeted corrective action plans. In this context, the complexity of the NGS techniques requires careful and continuous quality control procedures. We report a tumor BRCA1/2 (tBRCA) testing benchmark discrepancy provided by the European Molecular Genetics Quality Network in our laboratory during a round of EQA for somatic mutation testing of BRCA genes in relation to ovarian cancer. The critical analysis emerging from the tBRCA EQA is presented. We underline that harmonization processes are still required for the EQA in the molecular biology field, especially if applied to the evaluation of methods characterized by high complexity.
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Sequenciamento de Nucleotídeos em Larga Escala/normas , Neoplasias Ovarianas/genética , Proteína BRCA1/análise , Proteína BRCA1/genética , Proteína BRCA2/análise , Proteína BRCA2/genética , Benchmarking/métodos , Confiabilidade dos Dados , Feminino , Genes BRCA1 , Genes BRCA2 , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Laboratórios/normas , Controle de Qualidade , Reprodutibilidade dos TestesRESUMO
Recently, our lab, part of a referral center in Italy, reported its experience regarding the execution of germline BRCA1/2 (gBRCA) testing during the first months of the coronavirus disease-2019 (COVID-19) pandemic, which highlights a substantial reduction (about 60%) compared with the first 2 months of the current year. This evidence appeared to be a lockdown effect due to extraordinary restriction measures to slow down the spread of SARS-CoV-2. In this study, we aimed to evaluate the overall effects of the ongoing pandemic on gBRCA testing in our institution and to understand how COVID-19 has influenced testing after the complete lockdown (March 8-May 5, 2020). Additionally, we compared this year's trend with trends of the last 3 years to better monitor gBRCA testing progress. This detailed analysis highlights two important findings: (1) gBRCA testing did not increase significantly after the lockdown period (May-October 2020) compared with the lockdown period (March-April 2020), emphasizing that even after the lockdown period testing remained low. (2) Comparing the total tests per year (January-October 2017, 2018, 2019, with 2020), the impact of COVID-19 on gBRCA testing is apparent, with similarities of trends registered in 2017. These evidences reveal a gBRCA testing delay for cancer patients and healthy patients at this moment, and the new era of gBRCA testing in the management of ovarian, breast, pancreas and prostate cancer patients has been seriously questioned due to the COVID-19 pandemic. As consequence, we underline that measures to guarantee oncogenetic testing (e.g., gBRCA testing) along with new diagnostic/clinic strategies are mandatory. For these reasons, several proposals are presented in this study.
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Proteína BRCA1/sangue , Neoplasias da Mama/diagnóstico , COVID-19/epidemiologia , Neoplasias Ovarianas/diagnóstico , Neoplasias Pancreáticas/diagnóstico , Pandemias , Neoplasias da Próstata/diagnóstico , Biomarcadores Tumorais/sangue , COVID-19/psicologia , Diagnóstico Tardio/ética , Detecção Precoce de Câncer/estatística & dados numéricos , Feminino , Política de Saúde , Humanos , Itália/epidemiologia , Masculino , Distanciamento Físico , Quarentena/psicologia , SARS-CoV-2/patogenicidadeRESUMO
RCCX haplotypes with two copies of the CYP21A2 gene and one copy of the CYP21A1P pseudogene have been widely described in different populations. In most cases, the CYP21A2-like gene downstream of the TNXA gene showed a wild-type sequence or the c.293-13A/C > G variant while the CYP21A2 gene next to TNXB carried the p.(Gln319Ter) variant. Here is the discovery of a novel rare CYP21A2 haplotypes detected in an Italian patient with Non Classical Congenital Adrenal Hyperplasia (NC-CAH). The molecular family study was performed clarifying the previously found phenotype-genotype discrepancy.
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Hiperplasia Suprarrenal Congênita/genética , Esteroide 21-Hidroxilase/genética , Hiperplasia Suprarrenal Congênita/metabolismo , Hiperplasia Suprarrenal Congênita/fisiopatologia , Adulto , Alelos , Criança , Família , Feminino , Genótipo , Haplótipos/genética , Humanos , Masculino , Mutação , Fenótipo , Esteroide 21-Hidroxilase/metabolismoRESUMO
The first person-to-person transmission of the 2019-novel coronavirus in Italy on 21 February 2020 led to an infection chain that represents one of the largest known COVID-19 outbreaks outside Asia. Hospitals have been forced to reorganized their units in response to prepare for an unforeseen healthcare emergency. In this context, our laboratory (Molecular and Genomic Diagnostics Unit, Fondazione Policlinico Universitario Agostino Gemelli IRCCS) re-modulated its priorities by temporarily interrupting most of the molecular tests guaranteeing only those considered "urgent" and not postponable. In particular, this paper details changes regarding the execution of germline BRCA (gBRCA) testing in our laboratory. A substantial reduction in gBRCA testing (about 60%) compared to the first 2 months of the current year was registered, but the requests have not been reset. The requesting physicians were mainly gynaecologists and oncologists. These evidences further emphasize the new era of gBRCA testing in the management of cancer patients and confirms definitively the integration of gBRCA testing/Next Generation Sequencing (NGS) into clinical oncology. Finally, a re-organization of gBRCA testing in our Unit, mainly related to delayed and reduced arrival of tests was necessary, ensuring, however, a high-quality standard and reliability, mandatory for gBRCA testing in a clinical setting.
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Proteína BRCA2/genética , Neoplasias da Mama/diagnóstico , Infecções por Coronavirus/epidemiologia , Detecção Precoce de Câncer/estatística & dados numéricos , Neoplasias Ovarianas/diagnóstico , Pandemias , Pneumonia Viral/epidemiologia , Betacoronavirus/genética , Betacoronavirus/patogenicidade , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , COVID-19 , Teste para COVID-19 , Técnicas de Laboratório Clínico/estatística & dados numéricos , Infecções por Coronavirus/sangue , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/patologia , Detecção Precoce de Câncer/métodos , Diagnóstico Precoce , Feminino , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/estatística & dados numéricos , Humanos , Itália/epidemiologia , Mutação , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Pneumonia Viral/sangue , Pneumonia Viral/diagnóstico , Pneumonia Viral/patologia , Encaminhamento e Consulta/estatística & dados numéricos , SARS-CoV-2RESUMO
The multiple endocrine neoplasia type 1 (MEN1) is a rare syndrome characterized by the predisposition to developing multiple endocrine and non-endocrine tumors, typically characterized by the association between parathyroid gland hyperplasia or tumors, gastroenteropancreatic tumors and pituitary adenomas. The MEN1 gene is located on the long arm of chromosome 11 (11q13) and it encodes for the protein "menin". We here reported the case of a MEN1-patient, affected by primary hyperparathyroidism, insulinoma, pituitary non-hyperfunctioning adenoma and bilateral adrenal masses, carrying a novel heterozygous pathogenic variant (c.1252_1254delGACinsAT), located in exon 9 of MEN1 gene.
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Sequência de Bases , Éxons , Neoplasia Endócrina Múltipla Tipo 1/genética , Proteínas Proto-Oncogênicas/genética , Deleção de Sequência , Adulto , Humanos , MasculinoRESUMO
Correct classification of genomic variants causing potentially aberrant splicing is of utmost importance for patient management, especially in clinically actionable genes such as BRCA1/2. In this article, we report molecular evaluation of the BRCA1 c.439T>C (rs794727800, p.Leu147=) variant based on RNA of a patient suffering with high-grade serous ovarian cancer syndrome, to add new evidence to the only in silico data available for this variant. High Resolution Melting Analysis (HRMA) was used for the first time to investigate the spliceogenicity of a BRCA1 variant. HRMA with Sanger sequencing provided evidence that the c.439C allele does not cause aberrant splicing of the BRCA1 exon 7. In addition, HRMA with Sanger highlighted a different expression of the naturally occurring BRCA1 r.442_444del (c.442_444delCAG, p.Gln148del, at DNA level) isoform between blood and tumor, in this patient. HRMA is an alternative molecular approach to analyze spliceogenic properties of the c.439T>C variant and potentially for all those BRCA1/2 variants affecting splicing sites. These new evidences allowed to classify definitively the c.439T>C variant as benign. Furthermore, the different BRCA1 r.442_444del expression opens the discussion to consider a wider classification criteria for the splicing variants, including molecular evaluation at tissue level, which is an aspect currently scarcely considered in BRCA1/2 variant classification recommendations.
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Proteína BRCA1/genética , Desnaturação de Ácido Nucleico/genética , Polimorfismo de Nucleotídeo Único/genética , Splicing de RNA/genética , Idoso , Alelos , Proteína BRCA1/sangue , Sequência de Bases , DNA/genética , Feminino , HumanosRESUMO
The multifactorial likelihood analysis method has demonstrated utility for quantitative assessment of variant pathogenicity for multiple cancer syndrome genes. Independent data types currently incorporated in the model for assessing BRCA1 and BRCA2 variants include clinically calibrated prior probability of pathogenicity based on variant location and bioinformatic prediction of variant effect, co-segregation, family cancer history profile, co-occurrence with a pathogenic variant in the same gene, breast tumor pathology, and case-control information. Research and clinical data for multifactorial likelihood analysis were collated for 1,395 BRCA1/2 predominantly intronic and missense variants, enabling classification based on posterior probability of pathogenicity for 734 variants: 447 variants were classified as (likely) benign, and 94 as (likely) pathogenic; and 248 classifications were new or considerably altered relative to ClinVar submissions. Classifications were compared with information not yet included in the likelihood model, and evidence strengths aligned to those recommended for ACMG/AMP classification codes. Altered mRNA splicing or function relative to known nonpathogenic variant controls were moderately to strongly predictive of variant pathogenicity. Variant absence in population datasets provided supporting evidence for variant pathogenicity. These findings have direct relevance for BRCA1 and BRCA2 variant evaluation, and justify the need for gene-specific calibration of evidence types used for variant classification.
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Proteína BRCA1/genética , Proteína BRCA2/genética , Biologia Computacional/métodos , Mutação de Sentido Incorreto , Neoplasias/diagnóstico , Processamento Alternativo , Detecção Precoce de Câncer , Feminino , Predisposição Genética para Doença , Humanos , Funções Verossimilhança , Masculino , Herança Multifatorial , Neoplasias/genéticaRESUMO
The aim of this report is to describe results of BRCA1 and BRCA2 Next Generation Sequencing Analysis (NGS) analysis in 132 selected Italian patients with breast/ovarian cancer. A NGS pipeline with a reliable Copy Number Variation (CNV) prediction algorithm was applied. In addition, VarSome and Priors V2.0 Software were employed for in silico analysis of novel missense variants. A total of 37 BRCA1 and BRCA2 pathogenic variants were found in 34 unrelated subjects with a frequency of positive patients of 25.7% (34/132). Twenty-four deleterious variants were detected in BRCA1 (representing the 64.9% of all identified pathogenic defects) and thirteen (35.1% of all identified pathogenic variants) in BRCA2 gene. The percentage of patients carrying a variant of unknown significance (VUS) was 7.5% (10/132). In addition, seven novel variants (five in BRCA2 and two in BRCA1 gene), never previously reported, were identified. Our approach represents a robust and easy-to-use method for full BRCA1/2 screening. However, a consistent number of our high-risk families still remained without a satisfying answer. Necessarily, further collective efforts must be directed to a definitive classification of VUSs. The future auspice is that the use of multi-gene panel and more advanced screenings, such as whole exome sequencing and/or RNA seq, in routine diagnostics increases the detection rate.
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Neoplasias da Mama/genética , Genes BRCA1 , Genes BRCA2 , Estudos de Associação Genética , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Neoplasias Ovarianas/genética , Adulto , Idoso , Alelos , Substituição de Aminoácidos , Biomarcadores Tumorais , Neoplasias da Mama/diagnóstico , Estudos de Coortes , Biologia Computacional/métodos , Variações do Número de Cópias de DNA , Análise Mutacional de DNA , Feminino , Estudos de Associação Genética/métodos , Testes Genéticos , Genótipo , Mutação em Linhagem Germinativa , Humanos , Itália , Pessoa de Meia-Idade , Neoplasias Ovarianas/diagnóstico , LinhagemRESUMO
BRCA1/2 screening in Hereditary Breast and Ovarian Syndrome (HBOC) is an essential step for effective patients' management. Next-Generation Sequencing (NGS) can rapidly provide high throughput and reliable information about the qualitative and quantitative status of tumor-associated genes. Straightforwardly, bioinformatics methods play a key role in molecular diagnostics pipelines. BRCA1/2 genes were evaluated with our NGS workflow, coupled with Multiplex Amplicon Quantification (MAQ) and Multiplex Ligation-dependent Probe Amplification (MLPA) assays. Variant calling was performed on Amplicon Suite, while Copy Number Variant (CNV) prediction by in house and commercial CNV tools, before confirmatory MAQ/MLPA testing. The germline profile of BRCA genes revealed a unique HBOC pattern. Although variant calling analysis pinpointed heterozygote and homozygote polymorphisms on BRCA1 and BRCA2, respectively, the CNV predicted by our script suggested two conflicting interpretations: BRCA1 duplication and/or BRCA2 deletion. Our commercial software reported a BRCA1 duplication, in contrast with variant calling results. Finally, the MAQ/MLPA assays assessed a whole BRCA2 copy loss. In silico CNV analysis is a time and cost-saving procedure to powerfully identify possible Large Rearrangements using robust and efficient NGS pipelines. Our layout shows as bioinformatics algorithms alone cannot completely and correctly identify whole BRCA1/2 deletions/duplications. In particular, the complete deletion of an entire gene, like in our case, cannot be solved without alternative strategies as MLPA/MAQ. These findings support the crucial role of bioinformatics in deciphering pitfalls within NGS data analysis.
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Proteína BRCA2/genética , Deleção de Genes , Proteína BRCA1/genética , Feminino , Mutação em Linhagem Germinativa/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Pessoa de Meia-IdadeRESUMO
BACKGROUND: We report a novel BRCA1 LGR, involving the complete duplication of exon 3, in an Italian patient with a strong family history of breast and ovarian cancer. Our purpose is to provide an effective characterization of this LGR using a combination of different methods able to establish the exact breakpoints of the duplication. METHODS: MAQ assay was used as primary screening method in LGRs detection. Array CGH, RT-PCR, and Long-PCR were used for a careful characterization of rearrangement and breakpoint regions. The Repeat Masker program was employed to identify Alu sequences at breakpoint junctions. RESULTS: RNA analysis showed that this in tandem duplication of exon 3 causes an in frame insertion of 18 amino acids within the protein. Array CGH and Long-PCR strategies revealed that the duplication (g.100411_102863dup) involves exactly 2.452 nucleotides between intron 2 and intron 3 of the gene. In addition, while an Alu Sx sequence was identified at upstream breakpoint, no Alu repeats were found at downstream junction. This supports the hypothesis that the new duplication was the result of a non-homologous recombination event between Alu and Non-Alu sequences. CONCLUSION: Our strategy, which combines a comprehensive set of methodologies, has been able to characterize the new BRCA1 duplication confirming, as previously reported, that MAQ assay represents a reliable and effective method for a primary screening of BRCA rearrangements. We underline the relevance of incorporating quantitative methods for BRCA genes dosage testing into routine diagnostic practice.
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Proteína BRCA1/genética , Rearranjo Gênico , Síndrome Hereditária de Câncer de Mama e Ovário/genética , Elementos Alu , Pontos de Quebra do Cromossomo , Hibridização Genômica Comparativa , Feminino , Mutação em Linhagem Germinativa , Humanos , Itália , Pessoa de Meia-Idade , Linhagem , Análise de Sequência de DNARESUMO
BACKGROUND: Currently, multiplex ligation-dependent probe amplification (MLPA) is the most commonly used technique for the detection of large genomic rearrangements (LGRs) in the BRCA1/2 genes. However, a very fast assay, the BRCA1/2 multiplex amplicon quantification (MAQ), has been recently developed by Multiplicom. METHODS: As no data regarding the application of MAQ method to BRCA1/2 genes are available in literature, here we compared for the first time the performance of the MAQ assay with MLPA by using several positive BRCA1/2 LGRs DNA samples (previously tested by MLPA). RESULTS: MAQ method was able to detect all BRCA1/2 LGRs and no false-positive or -negative results were obtained in independent repetitive experiments. CONCLUSIONS: We can affirm that MAQ, as well as MLPA method, results to be valid and reproducible tools for molecular diagnostics and we are confident that this assay can be used for BRCA1/2 mutational screening as a fast and safe alternative to MLPA, particularly in the first line of analysis.
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Neoplasias da Mama/genética , Rearranjo Gênico/genética , Genes BRCA1/fisiologia , Neoplasias Ovarianas/genética , Adulto , Detecção Precoce de Câncer , Feminino , Genômica , Humanos , Pessoa de Meia-Idade , MutaçãoRESUMO
Deficiency of 11ß-hydroxylase (11ß-OHD) is the second most common cause of congenital adrenal hyperplasia (CAH), accounting for 0.2-8% of all cases. The disease is transmitted as an autosomal recessive trait and the underlying genetic causes of 11ß-OHD are primarily small pathogenic variants affecting the CYP11B1 gene coding the 11ß-hydroxylase enzyme. However, special events complicate the molecular diagnosis of 11ß-OHD such as an unequal crossing over between the CYP11B2 (coding aldosterone synthase enzyme) and CYP11B1 genes. The resulting allele contains a hybrid gene, with a CYP11B2 5'-end and a CYP11B1 3'-end, where the CYP11B1 gene is under the control of the CYP11B2 promoter and thus not responding to the adrenocorticotropin (ACTH) but to angiotensin II and K+. This leads a reduction of cortisol production in 11ß-OHD. In particular, CYP11B2/CYP11B1 chimeric genes can be distinguished into two groups depending on the breakpoint site: chimeras with breakpoint after the exon 5 of CYP11B2 preserve the aldosterone synthase activity, the others with breakpoint before exon 5 lose this function. In the last case, a more severe phenotype is expected. The aim of this review was to explore the setting of CYP11B2/CYP11B1 chimeras in 11ß-OHD, performing a careful review of clinical literature cases.
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Hiperplasia Suprarrenal Congênita , Esteroide 11-beta-Hidroxilase , Humanos , Esteroide 11-beta-Hidroxilase/genética , Citocromo P-450 CYP11B2/genética , Hiperplasia Suprarrenal Congênita/diagnóstico , Hiperplasia Suprarrenal Congênita/genética , Hidrocortisona , Oxigenases de Função MistaRESUMO
BACKGROUND: Dissection of genotype-phenotype relationships in hemophilia B (HB) is particularly relevant for challenging (mild HB) or for HB-associated but unclassified factor (F)IX missense variants. OBJECTIVE: To contribute elements to interpret unclassified HB-associated FIX missense variants by a multiple-level approach upon identification of a reported, but uncharacterized, FIX missense variant associated with mild HB. METHODS: Molecular modeling of wild-type and V92A FIX variants, expression studies in HEK293 cells with evaluation of protein (ELISA, western blotting) and activity (activated partial thromboplastin time-based/chromogenic assays) levels after recombinant expression, and multiple prediction tools. RESULTS: The F9(NM_000133.4):c.275T>C (p.V92A) variant was found in a mild HB patient (antigen, 45.4 U/dL; coagulant activity, 23.6 IU/dL; specific activity, 0.52). Newly generated molecular models showed alterations in Gla/EGF1-EGF2 domain conformation impacting Ca++ affinity and protein-protein interactions with activated factor XI (FXIa). Multitool analysis indicated a moderate impact on protein structure/function of the valine-to-alanine substitution, in accordance with patient and modeling data. Expression studies on the V92A variant showed a specific activity (0.49 ± 0.07; wild-type, 1.0 ± 0.1) recapitulating that of the natural variant, and pointed toward a moderate activation impairment as the main determinant underlying the p.V92A defect. The validated multitool approach, integrated with evidence-based data, was challenged on a panel (n = 9) of unclassified FIX missense variants, which resulted in inferred protein (secretion/function) outputs and HB severity. CONCLUSION: The rational integration of multitool and multiparameter analyses contributed elements to interpret genotype/phenotype relationships of unclassified FIX missense variants, with implications for diagnosis, management, and treatment of HB patients, and potentially translatable into other human disorders.
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BACKGROUND: To determine whether the functional effects of oral supplementation with Saffron, a natural compound that proved to be neuroprotective in early age-related macular degeneration, are influenced by complement factor H (CFH) and age-related maculopathy susceptibility 2 (ARMS2) risk genotypes. METHODS: Thirty-three early AMD patients, screened for CFH (rs1061170) and ARMS2 (rs10490924) polymorphisms and receiving Saffron oral supplementation (20 mg/day) over an average period of treatment of 11 months (range, 6-12), were longitudinally evaluated by clinical examination and focal electroretinogram (fERG)-derived macular (18°) flicker sensitivity estimate. fERG amplitude and macular sensitivity, the reciprocal value of the estimated fERG amplitude threshold, were the main outcome measures. RESULTS: After three months of supplementation, mean fERG amplitude and fERG sensitivity improved significantly when compared to baseline values (p < 0.01). These changes were stable throughout the follow-up period. No significant differences in clinical and fERG improvements were observed across different CFH or ARMS2 genotypes. CONCLUSIONS: The present results indicate that the functional effect of Saffron supplementation in individual AMD patients is not related to the major risk genotypes of disease.
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Crocus/química , Suplementos Nutricionais , Predisposição Genética para Doença , Degeneração Macular/tratamento farmacológico , Degeneração Macular/genética , Extratos Vegetais/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Fator H do Complemento/genética , Demografia , Eletrorretinografia , Feminino , Heterozigoto , Humanos , Degeneração Macular/fisiopatologia , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Proteínas/genética , Fatores de RiscoRESUMO
INTRODUCTION: Defects in the steroid 21-hydroxylase gene (CYP21A2) cause 21-hydroxylase deficiency (21OHD), the main cause of congenital adrenal hyperplasia (CAH). The disease shows a broad spectrum of clinical forms, ranging from severe or classical (salt wasting, SW, and simple virilizing, SV), to mild late onset or nonclassical (NC). 21OHD affects 1 in 15,000 in its severe classic form and 1 in 200-1000 in its mild NC form. There are many studies reporting the frequency of CYP21A2 pathogenic variants in different populations; however, few of them provide comprehensive information about Italian patients. Here, we present genetic results from a cohort of 245 unrelated Italian individuals with clinical diagnosis of CAH due to 21OHD. METHODS: A specific polymerase chain reaction (PCR) protocol combined with Sanger sequencing was used for CYP21A2 analysis. The multiplex ligation-dependent probe amplification (MLPA) assay was employed for copy number variation (CNV) determination. RESULTS: One hundred fourteen (46.5%) of the index cases had the NC form, 57 (23.3%) had the SV form, and 74 (30.2%) presented the SW form of the disease. The most prevalent variant found in NC patients was the p.Val282Leu (51.3%), while the most frequent variants in the classical form were p.Ile173Asn (8.6%) and c.293-13C>G (26.0%). In our study, the frequency of large rearrangements was 15.3%, with CAH-X alleles representing 40% of all DEL/CONV. In addition, 12 alleles carried rare variants, and 1 had a novel variant p.(Arg342Gln). We observed phenotype-genotype correlation in 94.7% of cases. A complete concordance was observed in Groups 0 (enzyme activity completely impaired) where all patients had the SW form as expected. In Group A (0-1% residual enzyme activity), 78.4% of patients had the anticipated SW form while 21.6% were diagnosed with the SV form. Within Group B (~ 2% residual enzyme activity), 93.4% of patients exhibited SV form and 6.5% SW disease. Finally, 92.6% and 7.4% of patients belonging to Group C (enzyme partially impaired to ~ 20-60% residual activity) exhibited NC and SV phenotypes, respectively. CONCLUSION: This work, representing a comprehensive genetic study, expanded the CYP21A2 variants spectrum of Italian patients with 21OHD and could be helpful in prenatal diagnosis and genetic counseling.
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Hiperplasia Suprarrenal Congênita , Humanos , Hiperplasia Suprarrenal Congênita/diagnóstico , Hiperplasia Suprarrenal Congênita/genética , Mutação , Variações do Número de Cópias de DNA , Esteroide 21-Hidroxilase/genética , Fenótipo , Reação em Cadeia da Polimerase Multiplex , GenótipoRESUMO
Leukodystrophies are a group of rare neurodegenerative disorders, usually presenting in infancy with a variable combination of cognitive, motor, and coordination impairment. Adult-onset cases are even more rare, often representing a diagnostic challenge even for experienced neurologists. Here, we present a case of a 44-year-old man with subacute and rapidly progressive spastic paraplegia, whose brain MRI revealed white matter abnormalities compatible with a diagnosis of leukodystrophy. We discuss how to apply a simplified diagnostic algorithm to distinguish acquired leukoencephalopathies from leukodystrophies and how to delve into the maze of genetic testing for white matter diseases. In our patient, we reached the diagnosis of a treatable disorder, whose early recognition is essential to prevent severe neurologic deterioration.
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Doenças Desmielinizantes , Leucoencefalopatias , Doenças por Armazenamento dos Lisossomos , Paraparesia Espástica , Adulto , Masculino , Humanos , Paraparesia Espástica/etiologia , Paraparesia Espástica/genética , Leucoencefalopatias/genética , Imageamento por Ressonância Magnética , Testes Genéticos , Doenças por Armazenamento dos Lisossomos/genética , Doenças Desmielinizantes/genética , Raciocínio ClínicoRESUMO
Next-generation sequencing (NGS) is nowadays commonly used for clinical purposes, and represents an efficient approach for the molecular diagnosis of familial hypercholesterolemia (FH). Although the dominant form of the disease is mostly due to the low-density lipoprotein receptor (LDLR) small-scale pathogenic variants, the copy number variations (CNVs) represent the underlying molecular defects in approximately 10% of FH cases. Here, we reported a novel large deletion in the LDLR gene involving exons 4-18, identified by the bioinformatic analysis of NGS data in an Italian family. A long PCR strategy was employed for the breakpoint region analysis where an insertion of six nucleotides (TTCACT) was found. Two Alu sequences, identified within intron 3 and exon 18, could underlie the identified rearrangement by a nonallelic homologous recombination (NAHR) mechanism. NGS proved to be an effective tool suitable for the identification of CNVs, together with small-scale alterations in the FH-related genes. For this purpose, the use and implementation of this cost-effective, efficient molecular approach meets the clinical need for personalized diagnosis in FH cases.