Maturity-onset diabetes of the young (MODY) is a form of monogenic diabetes with autosomal dominant inheritance. GCK -MODY and HNF1A -MODY are the prevalent subtypes. Currently, there is growing concern regarding the correct interpretation of molecular genetic findings. The American College of Medical Genetics and Genomics (ACMG) updated guidelines to interpret and classify molecular variants. This study aimed to determine the prevalence of MODY ( GCK / HNF1A ) in a large cohort of Brazilian families, to report variants related to phenotype, and to classify them according to ACMG guidelines. One hundred and nine probands were investigated, 45% with clinical suspicion of GCK -MODY and 55% with suspicion of HNF1A -MODY. Twenty-five different variants were identified in GCK gene (30 probands-61% of positivity), and 7 variants in HNF1A (10 probands-17% of positivity). Fourteen of them were novel (12- GCK /2- HNF1A ). ACMG guidelines were able to classify a large portion of variants as pathogenic (36%- GCK /86%- HNF1A ) and likely pathogenic (44%- GCK /14%- HNF1A ), with 16% (5/32) as uncertain significance. This allows us to determine the pathogenicity classification more efficiently, and also reinforces the suspected associations with the phenotype among novel variants.

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.

BackgroundMaturity‐onset diabetes of the young (MODY) is a form of monogenic diabetes with autosomal dominant inheritance. To date, mutations in 11 genes have been frequently associated with this phenotype. In Brazil, few cohorts have been screened for MODY, all using a candidate gene approach, with a high prevalence of undiagnosed cases (MODY‐X).MethodsWe conducted a next‐generation sequencing target panel (tNGS) study to investigate, for the first time, a Brazilian cohort of MODY patients with a negative prior genetic analysis. One hundred and two patients were selected, of which 26 had an initial clinical suspicion of MODY‐GCK and 76 were non‐GCK MODY.ResultsAfter excluding all benign and likely benign variants and variants of uncertain significance, we were able to assign a genetic cause for 12.7% (13/102) of the probands. Three rare MODY subtypes were identified (PDX1/NEUROD1/ABCC8), and eight variants had not been previously described/mapped in genomic databases. Important clinical findings were evidenced in some cases after genetic diagnosis, such as MODY‐PDX1/HNF1B.ConclusionA multiloci genetic approach allowed the identification of rare MODY subtypes, reducing the large percentage of MODY‐X in Brazilian cases and contributing to a better clinical, therapeutic, and prognostic characterization of these rare phenotypes.

Maturity-Onset Diabetes of the Young (MODY) type 4 or PDX1 -MODY is a rare form of monogenic diabetes caused by heterozygous variants in PDX1 . Pancreatic developmental anomalies related to PDX1 are reported only in neonatal diabetes cases. Here, we describe dorsal pancreatic agenesis in 2 patients with PDX1 -MODY. The proband presented with diabetes since 14 years of age and maintained regular glycemic control with low doses of basal insulin and detectable C-peptide levels after 38 years with diabetes. A diagnosis of MODY was suspected. Targeted next-generation sequencing identified a heterozygous variant in PDX1 : c.188delC/p.Pro63Argfs*60. Computed tomography revealed caudal pancreatic agenesis. Low fecal elastase indicated exocrine insufficiency. His son had impaired glucose tolerance, presented similar pancreatic agenesis, and harbored the same allelic variant. The unusual presentation in this Brazilian family enabled expansion upon a rare disease phenotype, demonstrating the possibility of detecting pancreatic malformation even in cases of PDX1 -related diabetes diagnosed after the first year of life. This finding can improve the management of MODY4 patients, leading to precocious investigation of pancreatic dysgenesis and exocrine dysfunction.

STUDY QUESTION Is there an (epi)genetic basis in patients with central precocious puberty (CPP) associated with multiple anomalies that unmasks underlying mechanisms or reveals novel genetic findings related to human pubertal control? SUMMARY ANSWER In a group of 36 patients with CPP associated with multiple phenotypes, pathogenic or likely pathogenic (epi)genetic defects were identified in 12 (33%) patients, providing insights into the genetics of human pubertal control. WHAT IS KNOWN ALREADY A few studies have described patients with CPP associated with multiple anomalies, but without making inferences on causalities of CPP. Genetic-molecular studies of syndromic cases may reveal disease genes or mechanisms, as the presentation of such patients likely indicates a genetic disorder. STUDY DESIGN, SIZE, DURATION This translational study was based on a genetic-molecular analysis, including genome-wide high throughput methodologies, for searching structural or sequence variants implicated in CPP and DNA methylation analysis of candidate regions. PARTICIPANTS/MATERIALS, SETTING, METHODS A cohort of 197 patients (188 girls) with CPP without structural brain lesions was submitted to a detailed clinical evaluation, allowing the selection of 36 unrelated patients (32 girls) with CPP associated with multiple anomalies. Pathogenic allelic variants of genes known to cause monogenic CPP (KISS1R, KISS1, MKRN3 and DLK1) had been excluded in the entire cohort (197 patients). All selected patients with CPP associated with multiple anomalies (n = 36) underwent methylation analysis of candidate regions and chromosomal microarray analysis. A subset (n = 9) underwent whole-exome sequencing, due to presenting familial CPP and/or severe congenital malformations and neurocognitive abnormalities. MAIN RESULTS AND THE ROLE OF CHANCE Among the 36 selected patients with CPP, the more prevalent associated anomalies were metabolic, growth and neurocognitive conditions. In 12 (33%) of them, rare genetic abnormalities were identified: six patients presented genetic defects in loci known to be involved with CPP (14q32.2 and 7q11.23), whereas the other six presented defects in candidate genes or regions. In detail, three patients presented hypomethylation of DLK1/MEG3:IG-DMR (14q32.2 disruption or Temple syndrome), resulting from epimutation (n = 1) or maternal uniparental disomy of chromosome 14 (n = 2). Seven patients presented pathogenic copy number variants: three with de novo 7q11.23 deletions (Williams–Beuren syndrome), three with inherited Xp22.33 deletions, and one with de novo 1p31.3 duplication. Exome sequencing revealed potential pathogenic variants in two patients: a sporadic female case with frameshift variants in TNRC6B and AREL1 and a familial male case with a missense substitution in UGT2B4 and a frameshift deletion in MKKS. LIMITATIONS, REASONS FOR CAUTION The selection of patients was based on a retrospective clinical characterization, lacking a longitudinal inclusion of consecutive patients. In addition, future studies are needed, showing the long-term (mainly reproductive) outcomes in the included patients, as most of them are not in adult life yet. WIDER IMPLICATIONS OF THE FINDINGS The results highlighted the relevance of an integrative clinical-genetic approach in the elucidation of mechanisms and factors involved in pubertal control. Chromosome 14q32.2 disruption indicated the loss of imprinting of DLK1 as a probable mechanism of CPP. Two other chromosomal regions (7q11.23 and Xp22.33) represented new candidate loci potentially involved in this disorder of pubertal timing. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by grant number 2018/03198-0 (to A.P.M.C.) and grant number 2013/08028-1 (to A.C.V.K) from the São Paulo Research Foundation (FAPESP), and grant number 403525/2016-0 (to A.C.L.) and grant number 302849/2015-7 (to A.C.L.) and grant number 141625/2016-3 (to A.C.V.K) from the National Council for Scientific and Technological Development (CNPq). The authors have nothing to disclose. TRIAL REGISTRATION NUMBER N/A.