Targeted massively parallel sequencing panel to diagnose genetic endocrine disorders in a tertiary hospital.

OBJECTIVES: To analyze the efficiency of a multigenic targeted massively parallel sequencing panel related to endocrine disorders for molecular diagnosis of patients assisted in a tertiary hospital involved in the training of medical faculty. MATERIAL AND METHODS: Retrospective analysis of the clinical diagnosis and genotype obtained from 272 patients in the Endocrine unit of a tertiary hospital was performed using a custom panel designed with 653 genes, most of them already associated with the phenotype (OMIM) and some candidate genes that englobes developmental, metabolic and adrenal diseases. The enriched DNA libraries were sequenced in NextSeq 500. Variants found were then classified according to ACMG/AMP criteria, with Varsome and InterVar. RESULTS: Three runs were performed; the mean coverage depth of the targeted regions in panel sequencing data was 249×, with at least 96.3% of the sequenced bases being covered more than 20-fold. The authors identified 66 LP/P variants (24%) and 27 VUS (10%). Considering the solved cases, 49 have developmental diseases, 12 have metabolic and 5 have adrenal diseases. CONCLUSION: The application of a multigenic panel aids the training of medical faculty in an academic hospital by showing the picture of the molecular pathways behind each disorder. This may be particularly helpful in developmental disease cases. A precise genetic etiology provides an improvement in understanding the disease, guides decisions about prevention or treatment, and allows genetic counseling.

A truncating variant of RAD51B associated with primary ovarian insufficiency provides insights into its meiotic and somatic functions.

Primary ovarian insufficiency (POI) causes female infertility by abolishing normal ovarian function. Although its genetic etiology has been extensively investigated, most POI cases remain unexplained. Using whole-exome sequencing, we identified a homozygous variant in RAD51B -(c.92delT) in two sisters with POI. In vitro studies revealed that this variant leads to translation reinitiation at methionine 64. Here, we show that this is a pathogenic hypomorphic variant in a mouse model. Rad51bc.92delT/c.92delT mice exhibited meiotic DNA repair defects due to RAD51 and HSF2BP/BMRE1 accumulation in the chromosome axes leading to a reduction in the number of crossovers. Interestingly, the interaction of RAD51B-c.92delT with RAD51C and with its newly identified interactors RAD51 and HELQ was abrogated or diminished. Repair of mitomycin-C-induced chromosomal aberrations was impaired in RAD51B/Rad51b-c.92delT human and mouse somatic cells in vitro and in explanted mouse bone marrow cells. Accordingly, Rad51b-c.92delT variant reduced replication fork progression of patient-derived lymphoblastoid cell lines and pluripotent reprogramming efficiency of primary mouse embryonic fibroblasts. Finally, Rad51bc.92delT/c.92delT mice displayed increased incidence of pituitary gland hyperplasia. These results provide new mechanistic insights into the role of RAD51B not only in meiosis but in the maintenance of somatic genome stability.

Severe Resistance to Thyroid Hormone Beta in a Patient with Athyreosis.

We report a patient with congenital hypothyroidism due to athyreosis complicated by a heterozygous thyroid hormone receptor beta (THRß) gene mutation (R320L), resulting in a severe resistance to thyroid hormone beta phenotype. The proband inherited the mutant allele from his father, presenting a very mild phenotype. While the precise reason for this discrepancy remains unknown, we postulate the possibility of de novo mutation and mosaicism in the father. Correlating thyrotropin (TSH) with free thyroxine (fT4) allowed us to predict the amount of fT4 required to normalize the proband's TSH, which supported the treatment with high dose of levothyroxine.

Targeted massively parallel sequencing panel to diagnose genetic endocrine disorders in a tertiary hospital

Abstract Objectives To analyze the efficiency of a multigenic targeted massively parallel sequencing panel related to endocrine disorders for molecular diagnosis of patients assisted in a tertiary hospital involved in the training of medical faculty. Material and methods Retrospective analysis of the clinical diagnosis and genotype obtained from 272 patients in the Endocrine unit of a tertiary hospital was performed using a custom panel designed with 653 genes, most of them already associated with the phenotype (OMIM) and some candidate genes that englobes developmental, metabolic and adrenal diseases. The enriched DNA libraries were sequenced in NextSeq 500. Variants found were then classified according to ACMG/AMP criteria, with Varsome and InterVar. Results Three runs were performed; the mean coverage depth of the targeted regions in panel sequencing data was 249×, with at least 96.3% of the sequenced bases being covered more than 20-fold. The authors identified 66 LP/P variants (24%) and 27 VUS (10%). Considering the solved cases, 49 have developmental diseases, 12 have metabolic and 5 have adrenal diseases. Conclusion The application of a multigenic panel aids the training of medical faculty in an academic hospital by showing the picture of the molecular pathways behind each disorder. This may be particularly helpful in developmental disease cases. A precise genetic etiology provides an improvement in understanding the disease, guides decisions about prevention or treatment, and allows genetic counseling.

Human Type 1 Iodothyronine Deiodinase (DIO1) Mutations Cause Abnormal Thyroid Hormone Metabolism.

Background: Iodothyronine deiodinase-1 (D1) selenoenzyme regulates the systemic supply of active thyroid hormone (TH). Transient decrease in D1 enzymatic activity is clinically relevant and adaptive in nonthyroidal illness such as fasting or acute illness. However, DIO1 gene defects have not been reported in humans. Methods: Genetic analysis was performed using whole-exome sequencing in members of two unrelated families presenting with abnormal serum thyroid function tests. Plasmid constructs containing the two pathogenic DIO1 variants were used for in vitro studies assessing the kinetics of their enzymatic activity. Thyroid function tests were measured in Dio1 heterozygous-null mice. Results: We report the novel identification and characterization of two missense DIO1 pathogenic variants (resulting in p.Asn94Lys and p.Met201Ile) in two unrelated families presenting with abnormal TH metabolism with elevated serum reverse triiodothyronine (rT3) levels and rT3/T3 ratios. These characteristic in vivo parameters are also present in Dio1 heterozygous-null mice. Kinetic studies of the resulting mutant D1 proteins demonstrate two- to threefold higher Km indicating lower substrate affinity and slower enzyme velocity. Conclusions: We report the identification and characterization of two missense DIO1 pathogenic variants identified in families with abnormal TH metabolism. This is the first demonstration of inherited D1 deficiency in humans.

Comprehensive Genetic Analysis of 128 Candidate Genes in a Cohort With Idiopathic, Severe, or Familial Osteoporosis.

CONTEXT: The genetic bases of osteoporosis (OP), a disorder with high heritability, are poorly understood at an individual level. Cases of idiopathic or familial OP have long puzzled clinicians as to whether an actionable genetic cause could be identified. OBJECTIVE: We performed a genetic analysis of 28 cases of idiopathic, severe, or familial osteoporosis using targeted massively parallel sequencing. DESIGN: Targeted sequencing of 128 candidate genes was performed using Illumina NextSeq. Variants of interest were confirmed by Sanger sequencing or SNP array. PATIENTS AND SETTING: Thirty-seven patients in an academic tertiary hospital participated (54% male; median age, 44 years; 86% with fractures), corresponding to 28 sporadic or familial cases. MAIN OUTCOME MEASURE: The identification of rare stop-gain, indel, splice site, copy-number, or nonsynonymous variants altering protein function. RESULTS: Altogether, we identified 28 variants of interest, but only 3 were classified as pathogenic or likely pathogenic variants: COL1A2 p.(Arg708Gln), WNT1 p.(Gly169Asp), and IDUA p.(His82Gln). An association of variants in different genes was found in 21% of cases, including a young woman with severe OP bearing WNT1, PLS3, and NOTCH2 variants. Among genes of uncertain significance analyzed, a potential additional line of evidence has arisen for GWAS candidates GPR68 and NBR1, warranting further studies. CONCLUSIONS: While we hope that continuing efforts to identify genetic predisposition to OP will lead to improved and personalized care in the future, the likelihood of identifying actionable pathogenic variants in intriguing cases of idiopathic or familial osteoporosis is seemingly low.

Screening of targeted panel genes in Brazilian patients with primary ovarian insufficiency.

Primary ovarian insufficiency (POI) is a heterogeneous disorder associated with several genes. The majority of cases are still unsolved. Our aim was to identify the molecular diagnosis of a Brazilian cohort with POI. Genetic analysis was performed using a customized panel of targeted massively parallel sequencing (TMPS) and the candidate variants were confirmed by Sanger sequencing. Additional copy number variation (CNV) analysis of TMPS samples was performed by CONTRA. Fifty women with POI (29 primary amenorrhea and 21 secondary amenorrhea) of unknown molecular diagnosis were included in this study, which was conducted in a tertiary referral center of clinical endocrinology. A genetic defect was obtained in 70% women with POI using the customized TMPS panel. Twenty-four pathogenic variants and two CNVs were found in 48% of POI women. Of these variants, 16 genes were identified as BMP8B, CPEB1, INSL3, MCM9, GDF9, UBR2, ATM, STAG3, BMP15, BMPR2, DAZL, PRDM1, FSHR, EIF4ENIF1, NOBOX, and GATA4. Moreover, a microdeletion and microduplication in the CPEB1 and SYCE1 genes, respectively, were also identified in two distinct patients. The genetic analysis of eleven patients was classified as variants of uncertain clinical significance whereas this group of patients harbored at least two variants in different genes. Thirteen patients had benign or no rare variants, and therefore the genetic etiology remained unclear. In conclusion, next-generation sequencing (NGS) is a highly effective approach to identify the genetic diagnoses of heterogenous disorders, such as POI. A molecular etiology allowed us to improve the disease knowledge, guide decisions about prevention or treatment, and allow familial counseling avoiding future comorbidities.

Nonautoimmune Hyperthyroidism Caused by a Somatic Mosaic GNAS Mutation Involving Part of the Thyroid Gland.

Nonautoimmune hyperthyroidism caused by activating mutations in the GNAS gene is a rare condition. In this study, we report a five-year-old girl diagnosed with nonautoimmune hyperthyroidism and tall stature harboring a somatic mosaic gain-of-function mutation in the GNAS gene (NM_080425.3: c.2530C>T;p.Arg844Cys previously reported as NM_000516.5:c.601C>T;p.Arg201Cys) and referred to thereafter as R201C, in three of four quadrants of the thyroid gland. Provision of a molecular diagnosis may avoid unnecessary complete ablation of the thyroid gland.

Transcriptomic Response to 1,25-Dihydroxyvitamin D in Human Fibroblasts with or without a Functional Vitamin D Receptor (VDR): Novel Target Genes and Insights into VDR Basal Transcriptional Activity.

The vitamin D receptor (VDR) mediates vitamin D actions beyond bone health. While VDR activation by 1,25-dihydroxyvitamin D (1,25D) leads to robust transcriptional regulation, less is known about VDR actions in the absence of 1,25D. We analyzed the transcriptomic response to 1,25D in fibroblasts bearing a severe homozygous hereditary vitamin D resistant rickets-related p.Arg30* VDR mutation (MUT) and in control fibroblasts (CO). Roughly 4.5% of the transcriptome was regulated by 1,25D in CO fibroblasts, while MUT cells without a functional VDR were insensitive to 1,25D. Novel VDR target genes identified in human fibroblasts included bone and cartilage factors CILP, EFNB2, and GALNT12. Vehicle-treated CO and MUT fibroblasts had strikingly different transcriptomes, suggesting basal VDR activity. Indeed, oppositional transcriptional effects in basal conditions versus after 1,25D activation were implied for a subset of target genes mostly involved with cell cycle. Cell proliferation assays corroborated this conjectured oppositional basal VDR activity, indicating that precise 1,25D dosage in target tissues might be essential for modulating vitamin D actions in human health.

Exome Sequencing Reveals the POLR3H Gene as a Novel Cause of Primary Ovarian Insufficiency.

CONTEXT: Primary ovarian insufficiency (POI) is a cause of female infertility. However, the genetic etiology of this disorder remains unknown in most patients with POI. OBJECTIVE: To investigate the genetic etiology of idiopathic POI. PATIENTS AND METHODS: We performed whole-exome sequencing of 11 families with idiopathic POI. To gain insights into the potential mechanisms associated with this mutation, we generated two mouse lines via clustered regularly interspaced short palindromic repeats/Cas9 technology. RESULTS: A pathogenic homozygous missense mutation (c.149A>G; p.Asp50Gly) in the POLR3H gene in two unrelated families was identified. Pathogenic mutations in this subunit have not been associated with human disorders. Loss-of-function Polr3h mutation in mice caused early embryonic lethality. Mice with homozygous point mutation (Polr3hD50G) were viable but showed delayed pubertal development, characterized by late first estrus or preputial separation. The Polr3hD50G female and male mice showed decreased fertility later in life, associated with small litter size and increased time to pregnancy or to impregnate a female. Polr3hD50G mice displayed decreased expression of ovarian Foxo3a and lower numbers of primary follicles. CONCLUSION: Our manuscript provides a case of POI caused by missense mutation in POLR3H, expanding the knowledge of molecular pathways of the ovarian function and human infertility. Screening of the POLR3H gene may elucidate POI cases without previously identified genetic causes, supporting approaches of genetic counseling.

Two rare loss-of-function variants in the STAG3 gene leading to primary ovarian insufficiency.

BACKGROUND/AIM: Primary ovarian insufficiency (POI) is characterized by primary or secondary amenorrhea, infertility, low estradiol levels, and increased gonadotropin levels. Most cases of POI remain unsolved even after exhaustive investigation. Here, we performed a targeted massively parallel sequencing to identify the genetic diagnosis of primary ovarian insufficiency (POI) in a Brazilian patient. PATIENT AND METHODS: An adopted 21-year-old Brazilian woman with isolated POI was selected. A custom SureSelectXT DNA target enrichment panel was designed and sequenced on an Illumina NextSeq 500 sequencer. The variants were confirmed using Sanger sequencing. RESULTS: Two rare heterozygous pathogenic variants in the STAG3 gene were identified in our patient. An unpublished 1-bp duplication c.291dupC (p.Asn98Glnfs*2) and one stop codon variant c.1950C > A (p.Tyr650*) were identified in the STAG3 gene. Both undescribed heterozygous variants were absent in the public databases [1000Genomes, Exome Aggregation Consortium (ExAC), National Heart, Lung, and Blood Institute Exome Variant Server (NHLBI/EVS), database of Single Nucleotide Polymorphisms (dbSNP), Genome Aggregation Database (gnomAD)], and Online Archive of Brazilian Mutations (ABraOM) databases. Moreover, neither heterozygous variants were found in 400 alleles from fertile Brazilian women screened by Sanger sequencing. The parents' DNA was not available to segregate these variants. CONCLUSION: Our results suggested that POI is caused by pathogenic compound heterozygous variants in the STAG3 gene, supporting the key role of the STAG3 gene in the etiology of primary ovarian insufficiency.

Central Precocious Puberty Caused by a Heterozygous Deletion in the MKRN3 Promoter Region.

CONTEXT: Loss-of-function mutations in the coding region of MKRN3, a maternally imprinted gene at chromosome 15q11.2, are a common cause of familial central precocious puberty (CPP). Whether MKRN3 alterations in regulatory regions can cause CPP has not been explored to date. We aimed to investigate potential pathogenic variants in the promoter region of MKRN3 in patients with idiopathic CPP. PATIENTS/METHODS: A cohort of 110 patients with idiopathic CPP was studied. Family history of precocious sexual development was present in 25%. Mutations in the coding region of MKRN3 were excluded in all patients. Genomic DNA was extracted from peripheral blood leukocytes, and 1,100 nucleotides (nt) of the 5'-regulatory region of MKRN3 were amplified and sequenced. Luciferase assays were performed in GT1-7 cells transiently transfected with plasmids containing mutated and wild-type MKRN3 promoter. RESULTS: We identified a rare heterozygous 4-nt deletion (c.-150_-147delTCAG; -38 to -41 nt upstream to the transcription start site) in the proximal promoter region of MKRN3 in a girl with CPP. In silico analysis predicted that this deletion would lead to the loss of a binding site for a downstream res-ponsive element antagonist modulator (DREAM), a potential transcription factor for MKRN3 and GNRH1 expression. Luciferase assays demonstrated a significant reduction of MKRN3 promoter activity in transfected cells with a c.-150_- 147delTCAG construct plasmid in both homozygous and heterozygous states when compared with cells transfected with the corresponding wild-type MKRN3 promoter region. CONCLUSION: A rare genetic alteration in the regulatory region of MKRN3 causes CPP.

A novel homozygous 1-bp deletion in the NOBOX gene in two Brazilian sisters with primary ovarian failure.

PURPOSE: Primary ovarian failure (POF) is characterized by amenorrhea, hypoestrogenism, and elevated gonadotropin levels in women leading to infertility under the age of 40 years. POF is a heterogeneous disease with different causes, and several genes have been associated with the POF phenotype. Thus, Whole-exome sequencing (WES) was performed in a consanguineous family with two sisters affected by POF. METHODS: All exons of both sisters were massively sequenced by WES, and the segregation was confirmed by Sanger sequencing. RESULTS: The novel homozygous c.1489delT variant in the NOBOX gene was identified in the two sisters with POF. Their parents were heterozygous carriers of this variant and, therefore, consistent with an autosomal recessive mode of inheritance. The c.1489delT NOBOX variant has not been previously reported in any public available databases (1000Genomes, 6500ESP/EVS, ExAC, and gnomAD). Furthermore, this variant was neither present in 387 Brazilian exomes control individuals nor in 200 fertile Brazilian women screened by Sanger sequencing. CONCLUSION: We report the first familial case of a novel homozygous NOBOX variant with an autosomal recessive mode of inheritance, thus allowing for a genetic diagnosis of primary ovarian failure.

A Novel Homozygous Missense FSHR Variant Associated with Hypergonadotropic Hypogonadism in Two Siblings from a Brazilian Family.

Hypergonadotropic hypogonadism (HH) is defined by increased gonadotropin levels in men and women. Primary ovarian failure (POF) is a form of female infertility characterized by amenorrhea, hypoestrogenism, and elevated gonadotropin levels in women under the age of 40 years. Although several genes have been associated with POF, its causative genes remain to be identified. Here, we used whole-exome sequencing (WES) to study a consanguineous family with a 46,XX girl and a 46,XY man affected by HH. All exons of both siblings and their parents were captured and massively sequenced by WES, and the candidate variant was confirmed by Sanger sequencing. A novel c.1298C>A;p.Ala433Asp missense variant of the follicle-stimulating hormone receptor (FSHR) gene was found in both affected siblings in a homozygous state and in their parents in a heterozygous state. This FSHR variant is not present in available databases (1000 Genomes and NHLBI/EVS) and Brazilian exome controls. Moreover, it is highly conserved and predicted as deleterious in all prediction sites analyzed. In conclusion, the novel homozygous FSHR variant observed in 2 siblings with HH can expand the spectrum of FSHR mutations in humans.

High Frequency of MKRN3 Mutations in Male Central Precocious Puberty Previously Classified as Idiopathic.

BACKGROUND/AIMS: Recently, loss-of-function mutations in the MKRN3 gene have been implicated in the etiology of familial central precocious puberty (CPP) in both sexes. We aimed to analyze the frequency of MKRN3 mutations in boys with CPP and to compare the clinical and hormonal features of boys with and without MKRN3 mutations. METHODS: This was a retrospective review of clinical, hormonal and genetic features of 20 male patients with idiopathic CPP evaluated at an academic medical center. The entire coding regions of MKRN3, KISS1 and KISS1R genes were sequenced. RESULTS: We studied 20 boys from 17 families with CPP. All of them had normal brain magnetic resonance imaging. Eight boys from 5 families harbored four distinct heterozygous MKRN3 mutations predicted to be deleterious for protein function, p.Ala162Glyfs*14, p.Arg213Glyfs*73, p.Arg328Cys and p.Arg365Ser. One boy carried a previously described KISS1-activating mutation (p.Pro74Ser). The frequency of MKRN3 mutations among these boys with idiopathic CPP was significantly higher than previously reported female data (40 vs. 6.4%, respectively, p < 0.001). Boys with MKRN3 mutations had typical clinical and hormonal features of CPP. Notably, they had later pubertal onset than boys without MKRN3 abnormalities (median age 8.2 vs. 7.0 years, respectively, p = 0.033). CONCLUSION: We demonstrated a high frequency of MKRN3 mutations in boys with CPP, previously classified as idiopathic, suggesting the importance of genetic analysis in this group. The boys with CPP due to MKRN3 mutations had classical features of CPP, but with puberty initiation at a borderline age.

Carbonyl cyanide m-chlorophenylhydrazone induced calcium signaling and activation of plasma membrane H(+)-ATPase in the yeast Saccharomyces cerevisiae.

The plasma membrane H(+)-ATPase from Saccharomyces cerevisiae is an enzyme that plays a very important role in the yeast physiology. The addition of protonophores, such as 2,4-dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), also triggers a clear in vivo activation of this enzyme. Here, we demonstrate that CCCP-induced activation of the plasma membrane H(+)-ATPase shares some similarities with the sugar-induced activation of the enzyme. Phospholipase C and protein kinase C activities are essential for this activation process while Gpa2p, a G protein involved in the glucose-induced activation of the ATPase, is not required. CCCP also induces a phospholipase C-dependent increase in intracellular calcium. Moreover, we show that the availability of extracellular calcium is required for CCCP stimulation of H(+)-ATPase, suggesting a possible connection between calcium signaling and activation of ATPase.