Three Cases of Carney-Stratakis Syndrome: A Genetically Heterogeneous Disease.

Carney-Stratakis syndrome (CSS) is an autosomal dominant rare syndrome, with incomplete penetrance, characterized by the association of paragangliomas and/or pheochromocytomas and gastrointestinal stromal tumors (GISTs). CSS is caused by germline heterozygous loss-of-function pathogenic variants (PVs) in the succinate dehydrogenase subunit genes (SDHB, SDHC, SDHD), with SDHB and SDHD being the most frequent. To date, only 2 germline SDHC PVs (c.43 C > T; c.405 + 1G > A) have been described in 3 patients with CSS. Three patients with CSS and very distinct clinical presentations are reported here: 1 caused by a germline SDHC large deletion and the others with metastatic GIST and negative genetic investigation for SDHx defects. Two cases (1 and 2) presented with pheochromocytoma (case 1 also with abdominal paraganglioma) and metastatic GIST. Although these 2 cases fulfilled the diagnostic criteria for CSS, the genetic investigation for SDHx PVs by next-generation sequencing and multiplex ligation-dependent probe amplification was negative. Case 3 had a large abdominal paraganglioma and a small low-grade GIST not associated with recurrence or metastasis. This case harbored a germline SDHC exon 3 deletion, not previously reported. In conclusion, CSS is a rare and morbid disease with distinct clinical presentations and genetic heterogeneity, which can contribute to underdiagnosis.

Evidence for a Founder Effect of SDHB Exon 1 Deletion in Brazilian Patients With Paraganglioma.

CONTEXT: Limited information is available concerning the genetic spectrum of pheochromocytoma and paraganglioma (PPGL) patients in South America. Germline SDHB large deletions are very rare worldwide, but most of the individuals harboring the SDHB exon 1 deletion originated from the Iberian Peninsula. OBJECTIVE: Our aim was to investigate the spectrum of SDHB genetic defects in a large cohort of Brazilian patients with PPGLs. METHODS: Genetic investigation of 155 index PPGL patients was performed by Sanger DNA sequencing, multiplex ligation-dependent probe amplification, and/or target next-generation sequencing panel. Common ancestrality was investigated by microsatellite genotyping with haplotype reconstruction, and analysis of deletion breakpoint. RESULTS: Among 155 index patients, heterozygous germline SDHB pathogenic or likely pathogenic variants were identified in 22 cases (14.2%). The heterozygous SDHB exon 1 complete deletion was the most frequent genetic defect in SDHB, identified in 8 out of 22 (36%) of patients. Haplotype analysis of 5 SDHB flanking microsatellite markers demonstrated a significant difference in haplotype frequencies in a case-control permutation test (P = 0.03). More precisely, 3 closer/informative microsatellites were shared by 6 out of 8 apparently unrelated cases (75%) (SDHB-GATA29A05-D1S2826-D1S2644 | SDHB-186-130-213), which was observed in only 1 chromosome (1/42) without SDHB exon 1 deletion (X2 = 29.43; P < 0.001). Moreover, all cases with SDHB exon 1 deletion had the same gene breakpoint pattern of a 15 678 bp deletion previously described in the Iberian Peninsula, indicating a common origin. CONCLUSION: The germline heterozygous SDHB exon 1 deletion was the most frequent genetic defect in the Brazilian PPGL cohort. Our findings demonstrated a founder effect for the SDHB exon 1 deletion in Brazilian patients with paragangliomas.

Pathogenesis of Primary Aldosteronism: Impact on Clinical Outcome.

Primary aldosteronism (PA) is the most common form of secondary arterial hypertension, with a prevalence of approximately 20% in patients with resistant hypertension. In the last decade, somatic pathogenic variants in KCNJ5, CACNA1D, ATP1A1 and ATP2B3 genes, which are involved in maintaining intracellular ionic homeostasis and cell membrane potential, were described in aldosterone-producing adenomas (aldosteronomas). All variants in these genes lead to the activation of calcium signaling, the major trigger for aldosterone production. Genetic causes of familial hyperaldosteronism have been expanded through the report of germline pathogenic variants in KCNJ5, CACNA1H and CLCN2 genes. Moreover, PDE2A and PDE3B variants were associated with bilateral PA and increased the spectrum of genetic etiologies of PA. Of great importance, the genetic investigation of adrenal lesions guided by the CYP11B2 staining strongly changed the landscape of somatic genetic findings of PA. Furthermore, CYP11B2 staining allowed the better characterization of the aldosterone-producing adrenal lesions in unilateral PA. Aldosterone production may occur from multiple sources, such as solitary aldosteronoma or aldosterone-producing nodule (classical histopathology) or clusters of autonomous aldosterone-producing cells without apparent neoplasia denominated aldosterone-producing micronodules (non-classical histopathology). Interestingly, KCNJ5 mutational status and classical histopathology of unilateral PA (aldosteronoma) have emerged as relevant predictors of clinical and biochemical outcome, respectively. In this review, we summarize the most recent advances in the pathogenesis of PA and discuss their impact on clinical outcome.

Targeted massively parallel sequencing for congenital generalized lipodystrophy.

OBJECTIVE: Our aim is to establish genetic diagnosis of congenital generalized lipodystrophy (CGL) using targeted massively parallel sequencing (MPS), also known as next-generation sequencing (NGS). METHODS: Nine unrelated individuals with a clinical diagnosis of CGL were recruited. We used a customized panel to capture genes related to genetic lipodystrophies. DNA libraries were generated, sequenced using the Illumina MiSeq, and bioinformatics analysis was performed. RESULTS: An accurate genetic diagnosis was stated for all nine patients. Four had pathogenic variants in AGPAT2 and three in BSCL2. Three large homozygous deletions in AGPAT2 were identified by copy-number variant analysis. CONCLUSION: Although we have found allelic variants in only 2 genes related to CGL, the panel was able to identify different variants including deletions that would have been missed by Sanger sequencing. We believe that MPS is a valuable tool for the genetic diagnosis of multi-genes related diseases, including CGL.

Phosphodiesterase 2A and 3B variants are associated with primary aldosteronism.

Familial primary aldosteronism (PA) is rare and mostly diagnosed in early-onset hypertension (HT). However, 'sporadic' bilateral adrenal hyperplasia (BAH) is the most frequent cause of PA and remains without genetic etiology in most cases. Our aim was to investigate new genetic defects associated with BAH and PA. We performed whole-exome sequencing (paired blood and adrenal tissue) in six patients with PA caused by BAH that underwent unilateral adrenalectomy. Additionally, we conducted functional studies in adrenal hyperplastic tissue and transfected cells to confirm the pathogenicity of the identified genetic variants. Rare germline variants in phosphodiesterase 2A (PDE2A) and 3B (PDE3B) genes were identified in three patients. The PDE2A heterozygous variant (p.Ile629Val) was identified in a patient with BAH and early-onset HT at 13 years of age. Two PDE3B heterozygous variants (p.Arg217Gln and p.Gly392Val) were identified in patients with BAH and HT diagnosed at 18 and 33 years of age, respectively. A strong PDE2A staining was found in all cases of BAH in zona glomerulosa and/or micronodules (that were also positive for CYP11B2). PKA activity in frozen tissue was significantly higher in BAH from patients harboring PDE2A and PDE3B variants. PDE2A and PDE3B variants significantly reduced protein expression in mutant transfected cells compared to WT. Interestingly, PDE2A and PDE3B variants increased SGK1 and SCNN1G/ENaCg at mRNA or protein levels. In conclusion, PDE2A and PDE3B variants were associated with PA caused by BAH. These novel genetic findings expand the spectrum of genetic etiologies of PA.

Germline mutation landscape of multiple endocrine neoplasia type 1 using full gene next-generation sequencing.

Background Loss-of-function germline MEN1 gene mutations account for 75-95% of patients with multiple endocrine neoplasia type 1 (MEN1). It has been postulated that mutations in non-coding regions of MEN1 might occur in some of the remaining patients; however, this hypothesis has not yet been fully investigated. Objective To sequence for the entire MEN1 including promoter, exons and introns in a large MEN1 cohort and determine the mutation profile. Methods and patients A target next-generation sequencing (tNGS) assay comprising 7.2 kb of the full MEN1 was developed to investigate germline mutations in 76 unrelated MEN1 probands (49 familial, 27 sporadic). tNGS results were validated by Sanger sequencing (SS), and multiplex ligation-dependent probe amplification (MLPA) assay was applied when no mutations were identifiable by both tNGS and SS. Results Germline MEN1 variants were verified in coding region and splicing sites of 57/76 patients (74%) by both tNGS and SS (100% reproducibility). Thirty-eight different pathogenic or likely pathogenic variants were identified, including 13 new and six recurrent variants. Three large deletions were detected by MLPA only. No mutation was detected in 16 patients. In untranslated, regulatory or in deep intronic MEN1 regions of the 76 MEN1 cases, no point or short indel pathogenic variants were found in untranslated, although 33 benign/likely benign and three new VUS variants were detected. Conclusions Our study documents that point or short indel mutations in non-coding regions of MEN1 are very rare events. Also, tNGS proved to be a highly effective technology for routine genetic MEN1 testing.