Next Generation Sequencing in Endocrine Practice

Home , Barakat syndrome, Leydig cell hypoplasia

Molecular Genetics and Metabolism 115 (2015) 61–71

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Molecular Genetics and Metabolism

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Minireview Next generation sequencing in endocrine practice

Gregory P. Forlenza a,1, Amy Calhoun b,1, Kenneth B. Beckman c, Tanya Halvorsen a, Elwaseila Hamdoun a, Heather Zierhut d, Kyriakie Sarafoglou a, Lynda E. Polgreen e, Bradley S. Miller a, Brandon Nathan a, Anna Petryk a,⁎ a Department of Pediatrics, Division of Pediatric Endocrinology, University of Minnesota Masonic Children's Hospital, Minneapolis, MN 55454, USA b Department of Pediatrics, Division of Genetics and Metabolism, University of Minnesota Masonic Children's Hospital, Minneapolis, MN 55454, USA c University of Minnesota Genomics Center, Minneapolis, MN 55455, USA d Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA e Division of Pediatric Endocrinology and Metabolism, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA 90502, USA article info abstract

Article history: With the completion of the Human Genome Project and advances in genomic sequencing technologies, the use of Received 1 May 2015 clinical molecular diagnostics has grown tremendously over the last decade. Next-generation sequencing (NGS) Accepted 2 May 2015 has overcome many of the practical roadblocks that had slowed the adoption of molecular testing for routine Available online 3 May 2015 clinical diagnosis. In endocrinology, targeted NGS now complements biochemical testing and imaging studies. The goal of this review is to provide clinicians with a guide to the application of NGS to genetic testing for endo- Keywords: crine conditions, by compiling a list of established gene mutations detectable by NGS, and highlighting key phe- Gene Mutation notypic features of these disorders. As we outline in this review, the clinical utility of NGS-based molecular testing Hypophosphatasia for endocrine disorders is very high. Identifying an exact genetic etiology improves understanding of the disease, Hormone provides clear explanation to families about the cause, and guides decisions about screening, prevention and/or Adrenal treatment. To illustrate this approach, a case of hypophosphatasia with a pathogenic mutation in the ALPL gene Vitamin D detected by NGS is presented. PTH © 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license Thyroid (http://creativecommons.org/licenses/by-nc-nd/4.0/). Gonad Pituitary

Contents

1. Introduction...... 62 2. Materialandmethods...... 62 2.1. Next-generationsequencing...... 62 2.2. Methodsofdataacquisition...... 62 3. Approachtodiseaseprocessandclinicalimplicationsofgenetictesting...... 62 3.1. Disordersofboneandmineralmetabolism...... 62 3.1.1. Acaseofhypophosphatasia...... 64 3.2. Adrenaldisorders...... 64 3.3. Gonadaldisorders ...... 64 3.4. Pituitaryandhypothalamicdiseases...... 64 3.5. Thyroiddisorders...... 66 3.6. Selectedsyndromeswithmultipleendocrinopathies...... 66 4. Discussion...... 69 4.1. Futureofnextgenerationsequencing...... 70 Conﬂictofinterest...... 70 Acknowledgments...... 70 References...... 70

⁎ Corresponding author at: University of Minnesota Masonic Children's Hospital, Pediatric Endocrinology, East Building Room MB671, 2450 Riverside Ave., Minneapolis, MN 55454, USA. E-mail address: [email protected] (A. Petryk). 1 These authors have contributed equally to this work.

http://dx.doi.org/10.1016/j.ymgme.2015.05.002 1096-7192/© 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 62 G.P. Forlenza et al. / Molecular Genetics and Metabolism 115 (2015) 61–71

1. Introduction University of Minnesota Genomics Center. Genomic DNA was extracted from the blood sample. Sequencing libraries were prepared and se- The diagnostic approach to endocrine diseases has traditionally been quence capture performed according to Illumina protocols utilizing based on a constellation of physical findings, biochemical testing, and the TruSight One Sequencing Panel, with one minor modification. imaging studies. With advances in genomics and sequencing technolo- DNA libraries from clinical samples were pooled for sequence capture gies, the role of genetic diagnosis in endocrinology has taken on greater in groups of 10 samples (9 clinical samples plus one control sample) importance. The etiology of endocrinopathies is multifactorial and, rather than pools of 12 samples, as recommended in the standard when genetically determined, is frequently multigenic [1]. Yet, a signif- Illumina protocol, in order to increase read depth per sample. The icant number of endocrine disorders demonstrate Mendelian transmis- enriched DNA libraries were sequenced on an Illumina HiSeq 2500 in- sion, suggesting disease-causing mutations. Identification of a mutation strument using paired-end 100-bp sequencing reads, generating can complement biochemical studies and provide diagnostic clues for ≥20 M reads (4 Gb) per sample. Raw sequencing reads were mapped early detection and treatment. to the reference genome using Burrows–Wheeler Alignment [2]. Historically, the use of molecular testing has been sparse, due to its Raw alignment files were realigned in the neighborhood of indels, and expense and technical difficulty. As recently as ten years ago, DNA se- recalibrated for base quality accuracy using the Genome Analysis Tool quencing for medical diagnosis was unusual, performed only when Kit (GATK) [3,4]. Point mutation and indel calls in exons and adjoining there was a very strong clinical rationale to seek a genetic diagnosis. intronic regions were made using the GATK Unified Genotyper. Variants Tests were difficult to obtain, turn-around was slow, and results were interpreted according to guidance issued by the American College were often difficult to interpret; many tests were only available on of Medical Genetics [6]. Known variants with a minor allele frequency a research basis. However, the transition from Sanger sequencing N0.01 (1%) in the 1000 genomes dataset are considered unlikely to be to high-throughput next-generation sequencing (NGS) technologies the cause of rare Mendelian phenotypes. Coverage is excellent under has dramatically decreased the costs and time involved in obtaining our protocol, with N20× coverage at 100% of the loci in ALPL, yielding high quality DNA sequence data. asensitivityofN95% and a specificity of N99% for all subtypes of Much of this transformation derives from the fact that NGS hypophosphatasia. methods allow clinicians to investigate candidate genes “in parallel” rather than “in series”. Due to its cost, Sanger sequencing typically 2.2. Methods of data acquisition involves a serial process of sequencing one candidate gene after an- other, starting with the highest probability genes and proceeding to The list of endocrine disorders and selected syndromes with lower-probability genes, in a process referred to as “a genetic odys- multiple endocrinopathies was limited to those that are due to mu- sey”. The genetic odyssey can involve months of frustrating and ex- tations detectable by NGS that are included in the TruSight One Se- pensive searching. In contrast, NGS – in which the cost of adding quencing Panel. Disorders of bone and mineral metabolism were additional genes to a sequencing panel is minimal – permits all limited to vitamin D deficiency and resistance, parathyroid dis- genes-of-interest to be sequenced simultaneously. The parallel na- eases, hypophosphatasia, and hypophosphatemic rickets due to el- ture of NGS therefore allows for the rapid query of multiple loci at evated FGF23 levels. Gonadal disorders were limited to those that once, including an analysis as broad as that of the entire exome or are caused by abnormal hormone synthesis or action (CYP21A2 mu- genome [2–4]. tations were excluded due to presence of pseudogenes). Inclusion In clinical practice, then, a managing physician can quickly analyze all of an extensive and a rapidly growing number of disorders of sex of the genes in a cellular pathway. It is worth noting that the benefits of development was deemed to be outside the scope of this review. this kind of parallel analysis offer more than an increase in speed and de- Diseases caused by chromosomal abnormalities, disorders of glu- crease in cost. Indeed, evidence is emerging that multiple “hits” (muta- cose and insulin metabolism, obesity, and lipid disorders were ex- tions) in the same pathway or in related pathways are important cluded. Individual diseases were cross-referenced against Online disease modifiers, even in disorders with classical Mendelian inheritance Mendelian Inheritance in Man (OMIM) database (http://omim. [5]. In other words, since a “genetic odyssey” using Sanger sequencing org) to provide gene and disease identifiers. Tables were constructed typically ends once a genetic “hit” is found, it risks returning an incom- and most salient clinical phenotypes were listed as a guide to clinical plete picture of diseases even when successful. The unbiased NGS-based presentation. approach, in contrast, suffers from no such ascertainment bias. NGS has been rapidly adopted in the molecular genetics community and is widely available for clinical testing. Due to the quickly decreasing 3. Approach to disease process and clinical implications of cost, marked increase in availability, and significant clinical utility, genetic testing molecular sequencing as a clinical diagnostic test is fast moving from the purview of academic medical geneticists into the realm of 3.1. Disorders of bone and mineral metabolism multispecialty clinical care. Targeted NGS promises to revolutionize the diagnosis of endocrine Patients with parathyroid or vitamin D disorders may come to med- conditions. However, the utilization of NGS is hampered by a number ical attention because of either acute symptoms such as hypocalcemic of factors, including uncertainty about the selection of genes that seizures or more chronic manifestations of hypo- or hypercalcemia, in- could be tested for presence of mutations. Thus, the goal of this review cluding paresthesias, renal calculi, weakness, anorexia, polydipsia or is to provide clinicians with a guide to NGS-based genetic testing for en- polyuria. Phenotypically, skeletal deformities (genu varum, epiphyseal docrine conditions. To illustrate this approach, a patient presenting with widening) may point to the diagnosis of rickets. Characteristic facial clinical features of hypophosphatasia who underwent targeted NGS of appearance (micrognathia, short palpebral fissures, prominent nose the ALPL gene is presented. with relatively deficient alae, smooth philtrum, small ears), and presence of congenital heart defects may trigger an evaluation for DiGeorge syn- 2. Material and methods drome. Short stature, obesity, and short forth metacarpals in a patient with an elevated parathyroid hormone (PTH) level and low calcium 2.1. Next-generation sequencing level raises a possibility of pseudohypoparathyroidism. Confirmatory biochemical testing includes measurement of calcium, phosphorus, PTH, vi- Targeted NGS of ALPL gene was performed at the University of tamin D, alkaline phosphatase and other related blood, urine, and Minnesota Medical Center, Molecular Diagnostics Laboratory and the imaging studies. G.P. Forlenza et al. / Molecular Genetics and Metabolism 115 (2015) 61–71 63

Table 1

Disorders of bone and mineral metabolism

Disease Gene H OMIM Phenotype

DiGeorge syndrome TBX1 AD 602054a Hypoparathyroidism, micrognathia, low-set ears, hypertelorism, 188400b congenital heart defects, thymic hypoplasia Hyperparathyroidism 1, familial isolated primary CDC73 AD 607393a Malaise, anorexia, pancreatitis, renal calculi, pathologic fractures (HRPT2) 145000b Hyperparathyroidism 2 (hyperparathyroidism-jaw tumor CDC73 AD 607393a Ossifying fibromas of the mandible and maxilla, renal lesions, recurrent syndrome) (HRPT2) 145001b pancreatitis Hyperparathyroidism, neonatal severe CASR AR/AD 601199a Failure to thrive, hypotonia, fractures of the ribs, respiratory distress 239200b Hypoparathyroidism, familial isolated GCM2 AR/AD 603716a Cataracts, intracerebral calcification on CT scan, tetany, seizures (GCMB) 146200b PTH AR/AD 168450a 146200b Hypoparathyroidism–retardation–dysmorphism TBCE AR 604934a IUGR, short stature, small hands and feet, microcephaly, micrognathia, syndrome (HRD; Sanjad–Sakati syndrome) 241410b long philtrum, low-set posteriorly rotated ears, deep-set eyes, thin lips Hypoparathyroidism, sensorineural deafness, and GATA3 AD 131320a Hypoparathyroidism, sensorineural deafness, renal dysplasia renal dysplasia (Barakat syndrome) 146255b Hypophosphatasia ALPL AR 171760a Infantile form: manifests within the first 6 months of life, FTT, rickets, rib 241500b fractures, lack of ossification, craniosynostosis, hypotonia, seizures, respiratory infections/failure ALPL AR 171760a Childhood form: premature deciduous tooth loss (less than 5 years of 241510b age), rickets, short stature ALPL AR/AD 171760a Adult form: recurrent fractures, particularly metatarsal stress fractures, 146300b calcium pyrophosphate arthropathy; odontohypophosphatasia: periodontitis Hypophosphatemic rickets DMP1 AR 600980a Rickets, early fusion of cranial sutures, nerve deafness, elevated FGF23 241520b level ENPP1 AR 173335a Hypophosphatemia, elevated FGF23 level 613312b FGF23 AD 605380a Short stature, tooth abscesses, bone pain, lower limb deformities, 193100b pseudofractures (adult-onset), generalized weakness (adult-onset), elevated FGF23 level PHEX X 300550a Short stature, frontal bossing, hearing loss, recurrent dental abscesses, 307800b bone pain, lower limb deformities, pseudofractures (adult-onset), elevated FGF23 level Idiopathic infantile hypercalcemia CYP24A1 AR 126065a Increased sensitivity to vitamin D, failure to thrive, nephrocalcinosis, 143880b hypotonia, febrile episodes Pseudohypoparathyroidism Type IA (Albright hereditary GNAS AD 139320a Short stature, obesity, round face, brachydactyly, subcutaneous osteodystrophy) (maternal) 103580b ossifications Pseudohypoparathyroidism, type IB STX16 AD 603666a Rarely brachydactyly, short metacarpals, obesity 603233b Pseudohypoparathyroidism, type IC GNAS AD 139320a Short stature, obesity, round face, low nasal bridge, short neck, 612462b brachydactyly, subcutaneous ossifications Pseudopseudohypoparathyroidism GNAS AD 139320a See Albright hereditary osteodystrophy (paternal) 612463b Vitamin D hydroxylation-deficient rickets, type 1A CYP27B1 AR 609506a Failure to thrive, frontal bossing, delayed tooth eruption, enlarged (1α-hydroxylase deficiency, vitamin D-dependent 264700b epiphyses, lower limb deformities rickets, type 1) Vitamin D hydroxylation-deficient rickets, type 1B CYP2R1 AR 608713a FTT, frontal bossing, delayed tooth eruption enlarged epiphyses, lower (25-hydroxylase deficiency) 600081b limb deformities Vitamin D-dependent rickets, type 2A VDR AR 601769a FTT, sensorineural deafness, delayed tooth eruption, alopecia, cutaneous (vitamin D resistant rickets) 277440b cysts

AD, autosomal dominant; AR, autosomal recessive; FTT, failure to thrive; H, inheritance; X, X-linked. a Gene ID. b Disease ID.

While biochemical testing and imaging studies help categorize the function mutation in VDR provides a genetic basis for the elevated level disorder into broader diagnostic groups, mutational analysis serves of 1,25-dihydroxyvitamin D level [9], ruling out an increased production not only as a confirmatory diagnostic study, but also brings in a greater of 1,25-dihydroxyvitamin D by monocytic cells in non-endocrine gran- level of diagnostic precision (Table 1). For example, because of its im- ulomatous or neoplastic conditions. Identification of CDC73 mutation al- plications for other endocrine systems, it is important to confirm a lows early diagnosis of other family members and prompt intervention diagnostic suspicion of Albright hereditary osteodystrophy by testing to prevent long-term consequences, such as renal calculi. In the case of for mutations in GNAS gene [7]. Because this disorder is due to post- the hyperparathyroidism-jaw tumor syndrome, confirmation of CDC73 zygotic somatic mutations, the ability of NGS sequencing to detect may alleviate the anxiety that the jaw tumor is of neoplastic nature, mosaicism (which is not detected by traditional Sanger sequencing) im- and guide further therapy [10]. Identification of mutations in patients proves the sensitivity of molecular testing at this locus [8].Knowingthat with frequent fractures, particularly infants, that indicate osteogenesis the mutation is present will prompt the health care provider to test for imperfecta or other underlying bone diseases (e.g. hypophosphatasia, other associated endocrinopathies, resulting from resistance to TSH, go- disorders of vitamin D metabolism) have important medical and legal nadotropins, GHRH, or antidiuretic hormone. Identification of a loss-of- implications, for example in cases of suspected child abuse [11]. 64 G.P. Forlenza et al. / Molecular Genetics and Metabolism 115 (2015) 61–71

NGS not only serves as a confirmatory test for the known mutations, confirm a diagnosis that requires intensive monitoring (autoimmune but may also identify new mutations, or improve our understanding of polyendocrinopathy syndrome) or allow early detection not possible the clinical implications of the known pathogenic mutations. For exam- using biochemical testing alone. For example, X-linked adrenoleukodys- ple, hypophosphatasia (HPP) is a rare inherited disorder characterized trophy (a peroxisomal disorder affecting the adrenal cortex, the central by low level of tissue-nonspecific alkaline phosphatase (ALP) due to nervous system, and the testes) is confirmed biochemically by finding loss-of-function mutations in the ALPL gene. The disease has a wide elevated very long-chain fatty acids [15]. However, heterozygous fe- spectrum of clinical presentation, including (from the most to the least se- males may have normal levels of very long-chain fatty acids and vere) the perinatal (usually lethal), infantile, childhood and adult forms. only detection of a mutation in ABCD1 gene [16] can reliably estab- p.His381Arg mutation (previously referred to as p.H364R) has been re- lish heterozygosity, which has significant implications for the off- ported in conjunction with a second missense ALPL mutation in one spring of a carrier and siblings of the proband. Early diagnosis of case of a lethal autosomal recessive hypophosphatasia [12]. The case ALD prompts evaluation of adrenal function pre-symptomatically below illustrates that it can also be associated with a mild, childhood and can prevent adrenal crisis. Early intervention with hematopoi- form of HPP. etic cell transplantation to halt cerebral manifestations can improve neurological outcome and survival [17].NGSmayalsobeusedtore- 3.1.1. A case of hypophosphatasia fine a diagnosis and allow selection of a precise treatment course The patient is a 3.9 years old boy who presented with premature loss (e.g. subtypes of congenital adrenal hyperplasia). In addition, the of primary teeth starting at 2.5 years of age. His growth has been normal ability to perform genetic testing based on a panel of genes associated (height and growth velocity at the 49th percentile). He began walking with a particular hormonal abnormality can also help to identify and at 14 months. He has had no history of fractures, gait abnormalities, thereby guide the management of rare disorders that might otherwise bone pain, or poor muscle tone. He had low ALP level (59 U/L, be difficult to recognize. normal range 110–320), elevated urine phosphoethanolamine (350 nmol/mg creatinine, normal range 18–150), markedly elevated 3.3. Gonadal disorders pyridoxal 5'-phosphate (641 nmol/L, normal range 20–125), normal calcium, phosphorus, and 25-hydroxyvitamin D levels, mildly Abnormal sex hormone synthesis or action may be suspected in in- elevated 1,25-dihydroxyvitamin D level (80 pg/mL, normal range fancy or even prenatally on the basis of abnormal or ambiguous genita- 15–75), low iPTH level (4 pg/mL, normal range 12–72), and no evi- lia, or a mismatch between sex phenotype and karyotype obtained for dence of rickets on a hand radiograph. NGS identified a heterozy- other reasons. In other cases, diagnosis may be delayed until much gous missense mutation c.1142ANG (p.His381Arg) in ALPL, later when puberty does not progress normally, or even later when pre- indicating that this mutation can occur not only in a lethal autoso- mature menopause or fertility problems arise. In general, diagnosis is mal recessive HPP, but also a mild, presumably autosomal dominant, challenging as there is a great deal of phenotypic overlap between dif- form due to haploinsufficiency. His mother also has a low ALP level ferent gonadal disorders. Traditionally, the first step is a karyotype for (29 U/L, normal range 40–150), but has no history of fractures, bone genetic sex determination followed by a combination of imaging and pain, or premature loss of teeth. His father has a normal ALP level. hormonal studies. Even after all of these steps, many of these disorders Maternal great-grandmother had rickets in her youth. Paternal are simply categorized in a descriptive fashion that, although useful for grandmother started wearing dentures between the age of 40 and treatment, may leave families and practitioners with uncertainty about 50 years. Molecular analysis of ALPL in other family members has future sexual development, gender identity, fertility and the possible not been performed. emergence of associated comorbidities. Genotypic characterization of patients with gonadal disorders is 3.2. Adrenal disorders valuable, providing key information about prognosis and the need for screening for associated comorbidities, and guiding the nature or timing Endocrine diseases of the adrenal gland occur as a result of hyper- or of therapeutic interventions (Table 3). For example, subjects with muta- hypofunction of one or more layers of the adrenal cortex. Congenital de- tions in WT1 would require ongoing assessment of kidney function, and, fects may be suspected prenatally or at birth on the basis of physical depending on the specific mutation, may need to be screened for Wilms findings (virilization of XX female infants), as a result of newborn tumor [18]. In other cases, genotypic characterization may provide key screening (congenital adrenal hyperplasia) or symptomatic electro- information about the anticipated impact of puberty (e.g. likelihood of lyte abnormalities (hypoglycemia, hyponatremic seizures). In other virilization at puberty with HSD17B3 [19] mutations that may influence cases they may present later in childhood as symptoms manifest choice of gender-rearing or whether to remove or relocate ectopic over time (precocious pubertal changes, cushingoid features), or gonads. are unmasked by other illnesses or events (adrenal crises). The evaluation that follows differs depending on what hormonal derange- 3.4. Pituitary and hypothalamic diseases ment is first detected and whether it is deficient or in excess, but in each case the clinician must differentiate primary adrenal disease The clinical phenotype for patients with combined pituitary hor- from other disorders affecting adrenal pathways (e.g. pituitary/ mone deficiencies tends to include characteristic facial features with hypothalamic dysregulation in central hypocortisolism or down- prominent foreheads, midface hypoplasia, depressed nasal bridges, stream receptor defects in pseudohypoaldosteronism). Once this is and short noses with anteverted nostrils. The presence of these fea- established, a more extensive investigation for associated comorbid- tures would prompt a workup for pituitary hormone deficiencies ities often ensues. In some circumstances it may be difficult to dif- and, based on the identified hormone anomalies, lead to confirmato- ferentiate organic disease from transient, functional hormonal ry sequencing for the suspected gene such as PIT1 (POU1F1) [20] derangements, or iatrogenic causes as a result of prolonged gluco- (Table 4). corticoid treatment, leading to repeated ACTH stimulation testing NGS can be used in patients with pituitary anomalies to characterize and imaging studies. them by genotype as well as phenotype. Such characterization allows The ability to obtain targeted genetic information early in the diag- for prediction of future anomalies based on the understanding of nostic process provides valuable information with implications for diag- the molecular basis of embryonic pituitary development and the key nosis and treatment (Table 2). For example, detection of a mutation in genes that affect the cellular differentiation of pituitary cells [20]. POMC [13] or TBX19 [14] would obviate the need for ongoing monitor- Genes such as HESX1, LHX3,andLHX4 affect immature pituitary progen- ing of other pituitary functions. In other cases, molecular diagnosis can itor cells and therefore would be expected to impact all 5 mature G.P. Forlenza et al. / Molecular Genetics and Metabolism 115 (2015) 61–71 65

Table 2

Adrenal disorders

Disease Gene H OMIM Phenotype

Cortisol deficiency Achalasia–Addisonianism–Alacrima AAAS AR 605378a Alacrima, achalasia, short stature, microcephaly, CNS abnormalities, adrenal insufficiency usually in syndrome; AAAS (Allgrove syndrome; Triple 231550b the 1st decade A) ACTH deficiency POMC U 176830a Obesity, adrenal insufficiency, red hair 609734b TBX19 AR 604614a Adrenal hypoplasia, fasting hypoglycemia 201400b Adrenal hypoplasia congenita NR0B1 X 300473a Primary adrenal insufficiency, HH (DAX1) 300200b Adrenoleukodystrophy ABCD1 X 300371a Adrenal insufficiency, CNS symptoms 300100b Congenital adrenal hyperplasia CYP11A1 AR 118485a M: external genitalia can range from female to undervirilized male; F: normal genitalia; both: can 613743b present with adrenal and salt wasting crises in the neonatal period CYP11B1 AR 610613a M: hyperpigmented scrotum, postnatal virilization; F: virilized external genitalia; both: HTN in 2/3 of 202010b the cases. Non-classic forms in both sexes may present later as premature adrenarche, growth acceleration, bone advancement, PCOS, menstrual irregularity, hirsutism, and infertility in females. CYP17A1 AR 609300a M: external genitalia can range from female to undervirilized male, lack of secondary sex 202110b characteristics; F: normal genitalia, primary amenorrhea, lack of secondary sex characteristics; Both: HTN, hypokalemia HSD3B2 AR 613890a M: external genitalia can range from female to undervirilized male; F: virilized external genitalia. 201810b Both can present with adrenal and salt wasting crises in the neonatal period. Non-classic forms in both sexes may present later as premature adrenarche, growth acceleration, bone advancement, PCOS, menstrual irregularity, hirsutism, and infertility in females. POR AR 124015a M: May be under-masculinized at birth 613571b F: Virilized genitalia at birth despite low to normal circulating androgens Both: impaired steroidogenesis, including cortisol; may present with or without Antley–Bixler syndrome (craniosynostosis, radiohumeral synostosis, and variable skeletal and visceral anomalies); maternal virilization may occur during pregnancy, with postpartum resolution Congenital lipoid adrenal hyperplasia (Lipoid STAR AR 600617a M: external genitalia can range from female to undervirilized male; F: normal genitalia, ovarian CAH, CLAH) 201710b cysts (may progress to torsion), premature ovarian failure; both: severe adrenal insufficiency with salt wasting with high mortality in infancy. Corticosteroid-binding globulin deficiency SERPINA6 AD 122500a Hypotension, fatigue; many patients asymptomatic AR 611489b Glucocorticoid deficiency 1 (GCCD1, Familial MC2R AR 607397a FTT, tall stature, hyperpigmentation, recurrent hypoglycemic episodes glucocorticoid deficiency-1, FGD1) (ACTHR) 202200b Glucocorticoid deficiency 2 (GCCD2) MRAP AR 609196a Recurrent hypoglycemia, hyperpigmentation 607398b Glucocorticoid deficiency 3 (GCCD3, Familial FGD3 AR %609197 Recurrent hypoglycemia, hyperpigmentation glucocorticoid deficiency 3, FGD3) Glucocorticoid resistance NR3C1 AD 138040a HTN; infertility, hirsutism, hypoglycemia, fatigue 615962b

Cortisol excess ACTH-independent macronodular adrenal GNAS1 U 139320a Adrenal (ACTH-independent) Cushing syndrome of adult onset (40–60 yr) hyperplasia (AIMAH) 219080b Primary pigmented nodular adrenocortical PRKAR1A AD 188830a Adrenal (ACTH-independent) Cushing syndrome, truncal obesity, round face, hypertension disease 610489b PDE11A AD 604961a 610475b PDE8B U 603390a 614190b

Isolated aldosterone deficiency Hypoaldosteronism, congenital, due to CMO I CYP11B2 AR 124080a Familial hyperreninemic hypoaldosteronism, FTT, growth retardation, hypotension, intermittent deficiency 203400b fever Hypoaldosteronism, congenital due to CMO II CYP11B2 AR 124080a FTT, growth retardation, hypotension deficiency 610600b Pseudohypoaldosteronism NR3C2 AD 600983a FTT, vomiting, diarrhea, poor feeding, metabolic acidosis; onset in infancy, improves with age, some 177735b asymptomatic

Hyperaldosteronism 614232a 218030b Glucocorticoid-remediable aldosteronism CYP11B1 AD 610613a Adrenal hyperplasia, HTN that is suppressible by glucocorticoid treatment 103900b

AD, autosomal dominant; AR, autosomal recessive; CNS, central nervous system; F, females; FTT, failure to thrive; H, inheritance; HH, hypogonadotropic hypogonadism; HTN, hypertension; M, males; PCOS, polycystic ovary syndrome; U, inheritance unclear or unknown; X, X-linked. %Molecular basis unknown. a Gene ID. b Disease ID. pituitary cell types and their resultant hormones. By contrast, PIT1 with identiﬁed PIT1 (POU1F1) mutations require screening for develop- (POU1F1) controls somatotrope, thyrotrope and lactotrope differentia- ment of new GH and TSH anomalies but not LH, FSH or ACTH/cortisol tion but not gonadotrope or corticotrope development. Thus, patients anomalies. 66 G.P. Forlenza et al. / Molecular Genetics and Metabolism 115 (2015) 61–71

Table 3

Gonadal disorders

Disease Gene H OMIM Phenotype

Androgen insensitivity AR X 313700a M: MAIS — normal genitalia, gynecomastia, infertility; PAIS — ambiguous genitalia with variable May be minimally affected (MAIS), 300068b degrees of virilization, sparse pubic hair, gynecomastia; CAIS — female body habitus and external partial (PAIS) or complete (CAIS) genitalia, tall stature, sparse pubic hair, absent facial hair, blind vaginal pouch, inguinal or abdominal testes, inguinal hernias F (carriers): normal genitalia, breast development and fertility; may have tall stature and sparse pubic hair Aromatase deficiency CYP19A1 AR 107910a M: eunuchoid body proportions, tall stature, excess adipose tissue; F: virilization, amenorrhea, 613546b absent breast development, cystic ovaries; Both: fetal and maternal virilization (acne and hirsutism in mother resolves after delivery) Aromatase excess syndrome CYP19A1 AD/AR/X 107910a M: gynecomastia, premature growth spurt, decreased adult stature; F: usually asymptomatic, may 139300b have macromastia, premature growth spurt, menstrual irregularity, short stature; Both: normal fertility Estrogen resistance ESR1 AR 133430a M: axillary acanthosis nigricans, early coronary artery disease, hyperinsulinemia and impaired 615363b glucose tolerance; F: primary amenorrhea, absent breast development with no response to oral estrogen, small uterus, large polycystic ovaries, severe acne; Both: osteopenia, delayed bone age with continued growth into adulthood 17β-Hydroxysteroid Dehydrogenase HSD17B3 AR 605573a M: undervirilized external genitalia at birth, inguinal testes, virilization at puberty, gynecomastia, Deficiency 264300b infertility, hypothyroidism; F: normal genitalia and breast development; possible association with pubertal-onset hirsuitism and PCOS Leydig cell hypoplasia (M) LHCGR AR 152790a M: type 1 — female external genitalia, absent development of male secondary sex characteristics; Luteinizing hormone resistance (F) 238320b type 2 — milder with range of genital abnormalities; F: amenorrhea, infertility McCune–Albright syndrome GNAS1 SM 139320a Peripheral precocious puberty, café au lait spots, polyostotic fibrous dysplasia, facial asymmetry, 174800b deafness, blindness, hyperthyroidism, hyperparathyroidism, Cushing syndrome, acromegaly, hyperprolactinemia, gastrointestinal polyps and pituitary adenomas. Premature ovarian failure FMR1 X 309550a Amenorrhea, osteoporosis 311360b DIAPH2 U 300108a 300511b POF1B U 300603a 300604b FOXL2 U 605597a 608996b BMP15 X 300247a 300510b NOBOX U 610934a 611548b FIGLA AD 608697a 612310b NR5A1 U 184757a 612964b Testotoxicosis, familial LHCGR AD 152790a Male-limited, gonadotropin independent, precocious puberty (usually by age 4 years) with rapid 176410b virilization, small testes, not suppressible by gonadotropin releasing hormone analogs Testotoxicosis with GNAS U 139320a Male-limited precocious puberty, see pseudohypoparathyroidism type 1a 176410b pseudohypoparathyroidism type 1a

AD, autosomal dominant; AR, autosomal recessive; F, females; H, inheritance; M, males; PCOS, polycystic ovary syndrome; SM, somatic mosaic; U, inheritance unclear or unknown; X, X-linked. a Gene ID. b Disease ID.

3.5. Thyroid disorders For example, mutations in GLIS3 result in central hypothyroidism but can also result in neonatal diabetes, congenital glaucoma, and The etiology for most cases of congenital hypothyroidism stems deafness [21,22]. Through the use of NGS, an infant with evidence from either primary defects in thyroid hormone synthesis and/or secre- for central hypothyroidism could be accurately and rapidly tested tion, and secondary abnormalities of the hypothalamic–pituitary– and any associated medical problems identified. Moreover, identifi- thyroidal axis that result in reduced thyrotrope activity. The advent of cation of mutations can also have direct impact on preventive and newborn screening programs to detect congenital hypothyroidism has therapeutic decisions. For example, confirmation of RET mutation drastically reduced unrecognized and untreated cases. The benefits of helps determine the timing of prophylactic early thyroidectomy early treatment in such infants are well documented and are a glowing [23] in addition to increasing awareness of associated disorders example of the utility and cost effective nature of screening programs. that patients have to be tested for as discussed earlier. Thus, in thy- However, the underlying etiology for hypothyroidism is often left unde- roid disease, NGS can provide valuable clinical data for diagnostic, termined. Moreover, “milder” cases of hypothyroidism are increasingly prognostic, and family genetic counseling purposes. recognized where modest TSH elevation may or may not have a genetic basis or be clinically relevant, leaving a clinician with uncertainty 3.6. Selected syndromes with multiple endocrinopathies whether treatment is indicated. Utilizing NGS to screen this population would create a stronger rationale for treatment or observation, and Many well-known syndromes involve multiple endocrinopathies would further reveal potential mutations in known thyroid associated as part of their characteristic phenotype. Often these features evolve genes (Table 5). at different times and in non-classical ways, making clinical diagno- In addition, several congenital thyroid disorders are associated sis of even well-known syndromes challenging. For many syndromic with other endocrinopathies that require screening and treatment. conditions, earlier genotypic identification holds prognostic and G.P. Forlenza et al. / Molecular Genetics and Metabolism 115 (2015) 61–71 67

Table 4

Pituitary and hypothalamic diseases

Disease Gene H OMIM Phenotype

Disorders of growth Growth hormone deficiency, isolated GH1 AR 139250a Puppet (baby doll) facies, hypoglycemia, sexual ateleiotic dwarfism, proportionate short stature, 262400b truncal obesity, delayed dentition, delayed bone age, high-pitched voice GH1 AR 139250a Short stature, delayed bone age, frontal bossing, truncal obesity 612781b GHRHR AR 139191a 612781b GH1 AD 139250a Short stature, truncal obesity, delayed dentition, delayed bone age 173100b Growth hormone insensitivity with STAT5B AR 604260a Combined GH insensitivity and immunodeficiency; physical appearance similar to Laron immunodeficiency 245590b syndrome. Growth hormone insensitivity syndrome GHR AR 600946a Marked short stature, childlike body proportions in adults, short limbs, hip degeneration, limited (Laron syndrome) 262500b elbow extensibility, small face, high-pitched voice Kowarski syndrome GH1 AR 139250a Short stature, delayed bone age, bioinactive GH 262650b

Panhypopituitarism Panhypopituitarism, X-Linked SOX3 X 313430a Panhypopituitarism, pituitary dwarfism 312000b Pituitary hormone deficiency, combined POU1F1 AR/AD 173110a Short stature, FTT, prominent forehead, midface hypoplasia, depressed nasal bridge, deep-set (PIT1) 613038b eyes, short nose with anteverted nostrils; mental retardation PROP1 AR 601538a Short stature, neonatal hypoglycemia, hypoglycemic seizures, midface hypoplasia 262600b LHX3 AR 600577a Short stature, sensorineural deafness, short neck with limited rotation, mental retardation, 221750b midface hypoplasia LHX4 AD 602146a Short stature, very small sella turcica, abnormal petrous bone, hypoglycemia, midface hypoplasia 262700b HESX1 AD/AR 601802a Septo-optic dysplasia, short stature, GH deficiency, hypoglycemia, optic nerve hypoplasia, absent 182230b septum pellucidum, absent corpus callosum, hypoplastic anterior pituitary, ectopic or absent posterior pituitary, midline forebrain defects, supernumerary digits, hypoplastic digits, psychomotor retardation OTX2 AD 600037a Short stature, pituitary hypoplasia with ectopic posterior pituitary, midface hypoplasia 613986b

Pubertal disorders FSH deﬁciency, isolated FSHB AR 136530a Primary amenorrhea 229070b Hypogonadotropic hypogonadism with or KAL1 X 300836a Absent or incomplete sexual maturation by the age of 18 years, cleft palate and sensorineural without anosmia (Kallmann syndrome) 308700b hearing loss with variable frequency, in the presence of anosmia, HH is typically called “Kallmann Hypogonadotropic hypogonadism with or FGFR1 AD 136350a syndrome” without anosmia 147950b PROKR2 AR/AD/X 607123a 244200b PROK2 AD 607002a 610628b CHD7 AD 608892a 612370b FGF8 AD 600483a 612702b GNRHR AD 138850a 146110b KISS1R AR/AD 604161a 614837b NSMF AD 608137a 614838b TAC3 AR 162330a 614839b TACR3 AR 162332a 614840b GNRH1 AR 152760a 614841b KISS1 AR 603286a 614842b WDR11 AD 606417a 614858b HS6ST1 AD 604846a 614880b Precocious puberty, central KISS1R AD 604161a Isosexual precocious puberty, reduced adult height 176400b MKRN3 AD 603856a 615346b

Other pituitary/hypothalamic disorders ACTH deﬁciency POMC U 176830a See “Adrenal disorders (cortisol deﬁciency)” 609734b

(continued on next page) 68 G.P. Forlenza et al. / Molecular Genetics and Metabolism 115 (2015) 61–71

Table 4 (continued)

Pituitary and hypothalamic diseases

Disease Gene H OMIM Phenotype

Other pituitary/hypothalamic disorders TBX19 AR 604614a See “Adrenal disorders (cortisol deﬁciency)” 201400b Diabetes insipidus AVP AD 192340a Hypertelorism, broad short nose, long philtrum, decreased bone mineral density 125700b

AD, autosomal dominant; AR, autosomal recessive; FTT, failure to thrive; GH, growth hormone; H, inheritance; HH, hypogonadotropic hypogonadism; U, inheritance unclear or unknown; X, X-linked. a Gene ID. b Disease ID.

management benefits. For conditions such as IPEX and CHARGE syn- genotypic identification allows for periodic monitoring for known drome (Table 6), genetic identification aids in establishing a sched- neoplastic conditions as well as screening and counseling for rela- ule for endocrine screening. For the multiple endocrine neoplasias, tives of the proband.

Table 5

Thyroid disorders

Disease Gene H OMIM Phenotype

Hyperthyroidism, nonautoimmune TSHR AD 603372a Activating mutation, low birth weight, tachycardia, goiter 609152b Hypothyroidism, athyroidal with spiky hair and cleft FOXE1 AR 602617a Kinky hair, cleft palate, choanal atresia, bifid epiglottis Palate (Bamforth–Lazarus syndrome) (TTF-2) 241850b POU1F1 AD/AR 173110a See “Hypopituitarism (combined pituitary hormone deficiency)” (PIT1) 613038b PROP1 AR 601538a See “Hypopituitarism (combined pituitary hormone deficiency)” 262600b Hypothyroidism, congenital with choreoathetosis, with NKX2-1 AD 600635a Brain–lung–thyroid syndrome: choreoathetosis, ataxia, hypotonia, respiratory distress or without pulmonary dysfunction (TTF-1) 610978b syndrome Hypothyroidism, congenital with neonatal diabetes GLIS3 AR 610192a Neonatal DM, congenital glaucoma, deafness mellitus 610199b Hypothyroidism, congenital, nongoitrous TSHR AR 603372a TSH resistance, variable severity ranging from asymptomatic to euthyroidism to 275200b hypothyroidism PAX8 AR/Sp 167415a Hypothermia, lethargy, umbilical hernia, hoarse cry, macroglossia 218700b TSHB AR 188540a Growth retardation, depressed nasal bridge, macroglossia, umbilical hernia, hoarse cry 275100b NKX2-5 AD 600584a Severe growth retardation, heart defects, kidney disease, liver disease 225250b THRA AD 190120a Growth retardation, macrocephaly, macroglossia, low resting heart rate, hypotonia 614450b Thyroid carcinoma NTRK1 AD 191315a Medullary thyroid carcinoma 155240b RET AD 164761a 155240b Thyroid dyshormonogenesis SLC5A5 AR 601843a Thyroid iodine accumulation defect, goiter, thyroid nodules, macroglossia, growth 274400b retardation TPO AR 606765a Defective oxidation and organification of iodide, goiter 274500b SLC26A4 AR 605646a Pendred syndrome, thyroid hormone organification defect, sensorineural deafness, 274600b vestibular function defect, cochlear malformation, goiter, thyroid carcinoma TG AR 188450a Thyroid hormone coupling defect with iodide trapping, goiter, thyroid cancer 274700b IYD AR 612025a Iodotyrosine deiodinase deficiency, goiter, growth retardation 274800b DUOXA2 AR 612772a Thyroglobulin synthesis defect, goiter 274900b DUOX2 AR 606759a Iodide organification defect 607200b Thyroid hormone resistance, generalized (GRTH) THRB AD 190160a Goiter, speech delay, attention deficit/hyperactivity disorder 188570b Thyroid hormone resistance, generalized (Refetoff THRB AR 190160a Goiter, deafness, stippled epiphyses, exophthalmos Syndrome, GRTH) 274300b Thyroid hormone resistance, selective pituitary (PRTH) THRB AD 190160a Selective pituitary resistance, hyperthyroidism 145650b T3 resistance (Allan–Herndon–Dudley syndrome, AHDS) SLC16A2 X 300095a Microcephaly, elongated face, bitemporal narrowing, hypotonia, developmental delay, 300523b involuntary movements, joint deformities

AD, autosomal dominant; AR, autosomal recessive; DM, diabetes mellitus; H, inheritance; Sp, sporadic inheritance; X, X-linked. a Gene ID. b Disease ID. G.P. Forlenza et al. / Molecular Genetics and Metabolism 115 (2015) 61–71 69

Table 6

Selected syndromes with multiple endocrinopathies

Disease Gene H OMIM Phenotype

Autoimmune AIRE AR 607358a Hypoparathyroidism, adrenal insufficiency, hypogonadism, DM, chronic mucocutaneous candidiasis, hepatitis, polyendocrinopathy syndrome AD 240300b pernicious anemia (APS1) Carney complex PRKAR1A AD 188830a Pigmented nodular adrenocortical disease, spotty skin pigmentation, myxomas of the heart, skin, breast, 160980b schwannomas, pituitary adenomas, Sertoli cell tumors. CHARGE syndrome CHD7 AD 608892a Short stature, GH deficiency, parathyroid hypoplasia, gonadotropin deficiency, hypothyroidism, microcephaly, 214800b square face, micrognathia, facial asymmetry, small ears, deafness, colobomas, ptosis, hypertelorism, downslanting SEMA3E AD 608166a palpebral fissures, posterior choanal atresia, cleft lip/palate. 214800b Culler–Jones Syndrome GLI2 AD 165230a Short stature, midface hypoplasia, micropenis, polydactyly, developmental delay, panhypopituitarism 615849b Denys–Drash syndrome WT1 AD 607102a Males: undermasculinized (ambiguous) or female genitalia, gonadal dysgenesis with both ovarian and testicular 194080b tissue present, gonadoblastoma, Females: usually normal external genitalia and gonads; gonadal dysgenesis, gonadoblastoma and Wolffian structures have been reported Both: nephropathy, HTN, high risk (~90%) of nephroblastoma (Wilms tumor) Frasier syndrome WT1 AD 607102a Males: female external genitalia 136680b Females: normal external genitalia, primary amenorrhea Both: streak gonads, gonadoblastoma, glomerulopathy IPEX FOXP3 X 300292a Hypothyroidism, DM, enteropathy, immunodeficiency 304790b Kenney–Caffey syndrome TBCE AR 604934a Hypoparathyroidism, IUGR, short stature, delayed anterior fontanel closure, small hand and feet 244460b Microphthalmia, syndromic SOX2 AD 184429a Short stature, growth failure, microcephaly, frontal bossing, hearing loss, microphthalmia, anophthalmia, optic 206900b nerve hypoplasia, coloboma, VSD, PDA, rib abnormalities, esophageal atresia, vertebral anomalies, developmental delay Multiple endocrine neoplasia MEN1 AD 613733a Hyperparathyroidism, tumors of the pancreas and anterior pituitary, facial angiofibroma, lipomas, gingival type 1 (MEN1) 131100b papules Multiple endocrine neoplasia RET AD 164761a Hyperparathyroidism, medullary carcinoma of the thyroid, pheochromocytoma, cutaneous lichen amyloidosis type 2A (MEN2A) 171400b Multiple endocrine neoplasia RET AD 164761a Marfanoid habitus, medullary carcinoma of the thyroid, pheochromocytoma, mucosal neuromas type 2B (MEN2B) 162300b MEN4 CDKN1B AD 600778a Acromegaly, pituitary adenoma, renal angiomyolipoma 610755b Prader–Willi-like syndrome MAGEL2 AD 605283a Prader–Willi-like syndrome; hypogonadism, hyperphagia, autistic features 615547b

AD, autosomal dominant; AR, autosomal recessive; DM, diabetes mellitus; H, inheritance; HTN, hypertension; IUGR, intrauterine growth retardation; X, X-linked. a Gene ID. b Disease ID.

4. Discussion correlations and aiding in the development of genotype-oriented screening guidelines as larger established genotype cohorts are identified with As a science, molecular genetics is only 62 years old, dating back to increased clinical use of this technology. Registry programs with subse- the description of the structure of DNA by Watson, Crick and Franklin quent genotype–phenotype correlations could be established to better in 1953 [24]; the number of chromosomes in a human cell was not de- understand the relevance and genetic nature of endocrinopathies. termined until 1956 [25]. The study of DNA for diagnosis began with the Although interest continues to increase in the clinical use of whole- discovery by Dr. Jerome Lejune in 1959 that Down syndrome was exome sequencing or even whole-genome sequencing, routine use of caused by an extra copy of chromosome 21 [25]. In the half-century this technology is not recommended [28]. Systematic targeted NGS test- since, clinical molecular diagnostics have “exploded.” The Human ing of well-crafted panels avoids the major ethical and logistical pitfalls Genome Project took 13 years and was completed in 2003 [26].In associated with incidentally discovered findings in genes unrelated to 2015, clinical laboratories are able to return the same amount of data the patient's presenting problem [29].Furthermore,targetedNGS in a matter of a few months. The technology that changed a 13-year in- panels often have demonstrably better sensitivity for mutations than ternational multisite collaborative endeavor into a simple blood test whole exome protocols [30]. that any licensed provider can order is NGS. At the turn of the 21st cen- Propagation of NGS technologies has led to greater integration of tury, a two-gene cancer risk Sanger sequencing based test cost about genetic counseling into endocrine practice. Genetic counselors assist $2,500 (personal communication). Now, a complete exome study cov- with education about inheritance, benefits and limitations to test- ering about 22,000 genes at a typical commercial laboratory is quoted ing, interpretation of risk and results, as well as identification of re- at around $5,000 [27]. Clearly, NGS has overcome the practical obstacles sources and research opportunities [31]. Genetic counselors have to the clinical use of routine molecular testing. traditionally worked in a diverse set of specialty clinics that have NGS increases the diagnostic precision of endocrine and other dis- counseled individuals with endocrinopathies, including pediatric orders, which is of importance not only to the health care providers, but (e.g. congenital adrenal hyperplasia), infertility (e.g. premature also to the families seeking answers and definitive diagnosis, particularly ovarian failure), and oncology (e.g. multiple endocrine neoplasias) when more than one family member is affected. Knowing the exact ge- clinics. Skills from these traditional roles are highly transferrable netic etiology improves understanding of the disease, provides clear ex- to an unlimited number of specialties including endocrinology. planation to the families about the cause, and guides the decision about Major issues addressed by genetic counseling for endocrinopathies screening, prevention and/or treatment of the patient and the family. include adequate informed consent and pre-test counseling, identifica- NGS also generates new information, enhancing genotype–phenotype tion of at risk family members, increased identification and cascade 70 G.P. Forlenza et al. / Molecular Genetics and Metabolism 115 (2015) 61–71 testing for individuals at risk, increased accuracy of result interpretation, References and better facilitation of the testing process and disclosure of results [32]. As costs of genetic testing decrease and genetic information evolves, [1] E. Amendola, P. De Luca, P.E. Macchia, D. Terracciano, A. Rosica, G. 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