Whole-exome Sequencing: Opportunities in Pediatric Endocrinology

Mark E Samuels; Caroline Hasselmann; Cheri L Deal; Johnny Deladoey; Guy Van Vliet

Personalized Medicine. 2014;11(1):63-78.

Pediatric endocrinology services see a wide variety of patients with diverse clinical symptoms, including disorders of growth, metabolism, bone and sexual development. Molecular diagnosis plays an important role in this branch of medicine. Traditional PCR-based Sanger sequencing is a mainstay format for molecular testing in pediatric cases despite its relatively high cost, but the large number of gene defects associated with the various endocrine disorders renders gene-by-gene testing increasingly unattractive. Using new high-throughput sequencing technologies, whole genomes, whole exomes or candidate-gene panels (targeted gene sequencing) can now be cost-effectively sequenced for endocrine patients. Based on our own recent experiences with exome sequencing in a research context, we describe the general clinical ascertainment of relevant pediatric endocrine patients, compare different formats for next-generation sequencing and provide examples. Our view is that protocols involving next-generation sequencing should now be considered as an appropriate component of routine clinical diagnosis for relevant patients.

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Table 1. Endocrine candidate genes.
Gene	OMIM ID	Disorder (nomenclature as per OMIM)	OMIM ID
Pituitary axis and relevant hypothalamic function
AVP	192340	Diabetes insipidus	125700
CHD7	608892	Hypogonadotropic hypogonadism 5 with or without anosmia	612370
FGFR1	136350	Hypogonadotropic hypogonadism 2 with or without anosmia	147950
FGF8	600483	Hypogonadotropic hypogonadism 6 with or without anosmia	612702
GH	139250	Growth hormone deficiency, isolated, type IA, IB, 2	262400, 612781, 173100
GHR	600946	Short stature	604271
GNRH1	152760	Hypogonadotropic hypogonadism 12 with or without anosmia	614841
GLI2	165230	Holoprosencephaly 9	610829
GNRHR	138850	Hypogonadotropic hypogonadism 7 with or without anosmia	146110
HESX1	601802	Growth hormone deficiency with pituitary anomalies, septo-optic dysplasia	182230
IGF1	147440	Growth retardation of intrauterine onset with deafness	608747
KAL1	300836	Hypogonadotropic hypogonadism 1 with or without anosmia	308700
KISS1R	604161	Hypogonadotropic hypogonadism 8 with or without anosmia	614837
LHX3	600577	Combined pituitary deficiency 3	221750
LHX4	602146	Combined pituitary deficiency 4	262700
MC1R	155555	Skin/hair/eye pigmentation 2, blond hair/fair skin	266300
MC2R	607397	Glucocorticoid deficiency due to ACTH unresponsiveness	202200
MC4R	155541	Obesity	601665
MRAP	609196	Glucocorticoid deficiency 2	607398
OTX2	600037	Combined pituitary deficiency 6	613986
PITX2	601542	Axenfeld–Rieger syndrome type 1	180500
POMC	176830	Obesity, adrenal insufficiency, and red hair due to POMC deficiency	609734
POU1F1 (PIT1)	173110	Combined pituitary deficiency 1	613038
PROK2	607002	Hypogonadotropic hypogonadism 4 with or without anosmia	610628
PROKR2	607123	Hypogonadotropic hypogonadism 3 with or without anosmia	244200
PROP1	601538	Combined pituitary deficiency 2	262600
SEMA3A	603961	Hypogonadotropic hypogonadism 16 with or without anosmia	614897
SHH	600725	Holoprosencephaly 3, pituitary anomalies	142945
SHOX	312865	Short stature, idiopathic, familial	300582
SOX2	184429	Optic nerve hypoplasia and abnormalities of the CNS	206900
SOX3	313430	Panhypopituitarism, X-linked	312000
TAC3	162330	Hypogonadotropic hypogonadism 10 with or without anosmia	614839
TACR3	162332	Hypogonadotropic hypogonadism 11 with or without anosmia	614840
TBX19 (TPIT)	604614	ACTH deficiency	201400
WDR11	606417	Hypogonadotropic hypogonadism 14 with or without anosmia	614858
Thyroid axis
DIO1	147892	No definitive human mutations	NA
DIO2	601413	No definitive human mutations	NA
DUOXA2	612772	Thyroid dyshormonogenesis 5	274900
DUOX2	606759	Thyroid dyshormonogenesis 6	607200
FOXE1	602617	Bamforth–Lazarus syndrome, hypothyroidism with facial dysmorphology	241850
GLIS3	610192	Diabetes mellitus, neonatal, with congenital hypothyroidism	610199
IGSF1	300137	Central hypothyroidism	300888
IYD	612025	Thyroid dyshormonogenesis 4	274800
NKX2-5	600584	Hypothyroidism, congenital nongoitrous, 5, congenital cardiac defects	225250
PAX8	167415	Hypothyroidism, congenital, due to thyroid dysgenesis or hypoplasia	218700
SECISBP2	607693	Thyroid hormone metabolism, abnormal	609698
SERPINA7 (TBG)	314200	Thyroxine-binding globulin deficiency	NA
SLC5A5	601843	Thyroid dyshormonogenesis 1	274400
SLC16A2	300095	Also known as MCT8, Allan–Herndon–Dudley syndrome	300523
SLC26A4	605646	Pendred syndrome	274600
TG	188450	Thyroid dyshormonogenesis 3	274700
THRA	190120	Hypothyroidism, congenital, nongoitrous, 6	614450
THRB	190160	Thyroid hormone resistance	188570
TPO	606765	Thyroid dyshormonogenesis 2A	274500
TSHB	188540	Hypothryoidism, congenital, nongoitrous 4	275100
TSHR	603372	Hypothyroidism, congenital, nongoitrous 1, hyperthyroidism, nonautoimmune	275200, 609152
TTR (TBPA)	176300	Amyloidosis	105210
Parathyroid axis
CASR	601119	Hypoparathyroidism, hyperparathyroidism	146200, 239200
FAM111A	615292	Kenny-Caffey syndrome, hypoparathyroidism	244460
GCM2 (GCMB)	603716	Hypoparathyroidism	146200
GNA11	139313	Hypocalciuric hypercalcemia 2	145981
GNAS (GNAS1)	139320	Pseudohypoparathyroidism	103580
PTH	168450	Hypoparathyroidism	146200
PTH1R	168468	Metaphyseal chondrodysplasia, Murk Jansen type	156400
TBX1	602054	DiGeorge syndrome, hypoparathyroidism	188400
TBCE	604934	Hypoparathyroidism–retardation–dysmorphism syndrome	241410
Adrenal axis (metabolism and sexual function)
AIRE	607358	Autoimmune polyendocrinopathy syndrome, type I, with or without reversible metaphyseal dysplasia (APECED)	240300
AKR1C2	600450	46XY sex reversal 8	614279
AR	313700	Androgen insensitivity	300068
CBX2	602770	46XY sex reversal 5	613080
CYP11A1	118485	Adrenal insufficiency with 46XY sex reversal, partial or complete	613743
CYP11B1	610613	Adrenal hyperplasia, congenital, due to 11-beta-hydroxylase deficiency, with hyperaldosteronism	202010
CYP17A1	609300	Adrenal hyperplasia, congenital, due to 17-alpha-hydroxylase deficiency	202110
CYP21A2	613815	Adrenal hyperplasia, congenital, due to 21-hydroxylase deficiency	201910
DHH	233420	46XY sex reversal 7	605423
EDNRA	131243	ACTH-independent macronodular adrenal hyperplasia	219080
GNAS	139320	ACTH-independent macronodular adrenal hyperplasia, more commonly McCune–Albright syndrome	219080, 174800
HSD3B2	613890	Adrenal hyperplasia with 3-beta-hydroxysteroid dehydrogenase, type II, deficiency	201810
KCNJ5	600734	Hyperaldosteronism, familial, type III	613677
MAP3K1	600982	46XY sex reversal 6	613762
NR5A1	184757	46XY sex reversal 3	612965
NR0B1 (DAX1)	300473	46XY sex reversal 2	300018
NNT	607878	Glucocorticoid deficiency 4, ACTH resistance, adrenal insufficiency, diabetes	614736
POR	124015	Antley–Bixler syndrome	201750
SOX3	313430	46XX sex reversal 3	300833
SOX9	608160	46XX sex reversal 2	278850
SRY	480000	46XY sex reversal 1, 46XX sex reversal 1	400044, 400045
STAR	600617	Lipoid adrenal hyperplasia	201710
WNT4	603490	Mullerian aplasia and hyperandrogenism, SERKAL syndrome	158330, 611812
Pancreas axis
GCK	138079	MODY, type 2	125851
HNF1A	142410	MODY, type 3	600496
HNF4A	600281	MODY, type 1	125850
INS	176730	MODY, type 10	613370
INSR	147670	Diabetes mellitus, insulin-resistant, with acanthosis nigricans, leprechaunism	610549, 246200
TCF7L2	602228	Diabetes mellitus, Type 2, susceptibility to	125853
Chromosomal abnormalities with endocrine phenotypes
45, XO	NA	Turner syndrome	NA
47, XXY	NA	Klinefelter syndrome	NA
15q11–q13 paternal deletion	NA	Prader–Willi syndrome (patients typically followed by endocrinology)	176270
15q11–q13 maternal deletion	NA	Angelman syndrome (patients not normally followed by endocrinology)	105830

This list is not intended to be comprehensive. It preferentially includes genes leading to direct or indirect endocrine hormone-related phenotypes, but does not include all syndromic genes in which mutations result in patients referred to Endocrinology Services. Genes are listed according to the main endocrine axis affected by mutations, not necessarily by the physical site of expression of the gene product as many of these genes are expressed in multiple tissues. Some genes mutate to multiple phenotypes, in which case those most directly relevant to endocrine function only are listed; see OMIM gene entries for additional details in all cases [7].
APECED: Autoimmune polyendocrinopathy, candidiasis, ectodermal dysplasia; MODY: Maturity onset diabetes of the young; NA: Not applicable; SERKAL: Sex reversal with dysgenesis of kidneys, adrenals and lungs.

Sidebar

Executive Summary

Clinical ascertainment in pediatric endocrinology

Pediatric endocrinology services in tertiary care hospitals are referred patients with a wide range of disorders affecting growth, sexual development and maturation and metabolism.
Relevant medical disorders include those of central dysfunction (hypothalamus and/or pituitary), peripheral dysfunction (thyroid, parathyroid, adrenals and/or pancreas) or general dysfunction (multiple nonendocrine tissues). Mutations are known to occur in many genes that affect one or more of these organ systems.

Classical molecular diagnostics

Currently, molecular diagnostics for endocrine disorders are usually performed on a gene-by-gene, patient-by-patient basis using PCR–Sanger fluorescent capillary sequencing. This technology is expensive and typically performed on candidate genes sequentially until all known genes are excluded, leading to clinicians being conservative in the number of genes tested and delays in return of results.

Next-generation sequencing

Next-generation sequencing, either whole-genome sequencing (WGS) or whole-exome sequencing (WES), is now routinely employed in human genetics research. Costs per gene sequenced are orders of magnitude less than for the equivalent amount of PCR–Sanger sequencing, and all genes are sequenced in the same procedure, and thus are completely parallelized. Next-generation sequencing does entail technical caveats, including incomplete coverage of some genes, and difficulties in aligning and mutation detection of sequences from duplicated gene families or segments. WES technology does not cover most intron or regulatory element sequences, although for now it is cost effective compared with WGS.

Examples from a pediatric endocrinology service

In a pilot research project we analyzed WES data for six families with patients of unexplained etiology. All six families were explained, with mutations either in known genes (with atypical clinical or laboratory presentations) or in two cases the same novel gene.

Conclusion

WES has demonstrated success in permitting diagnoses of patients with otherwise unexplained etiology, in both research and clinical contexts. For the moment, WGS remains too expensive for routine clinical uptake.
Regarding the transition to the use of exome sequencing in routine clinical context, major concerns are lack of technical standardization of the instrumentation and bioinformatic analysis software, and ethical issues arising from potentially actionable incidental findings in genes unrelated to endocrinology. Initiatives are underway in the international human genetics community to resolve these issues.
In the immediate term, targeted sequencing of endocrine-specific candidate genes, using the same basic technology as in WES, offers an attractive alternative minimizing some technical problems and eliminating the issue of incidental findings, as problematic genes such as oncogenes would normally be excluded.
Improved molecular diagnosis through high-throughput sequencing is certain to have a strongly positive impact on patient management, and has good potential to reduce overall costs to the healthcare system when averaged over many patients.