A young boy with ventricular arrhythmias and thyroid dysgenesis: two genes are not enough?

Abstract

Congenital hypothyroidism (CH) may be caused by biallelic variants in the TSHR gene. CH due to thyroid dysgenesis has also been linked to pathogenic variants of the nucleotide kinase 2, homeobox 5 (NKX2-5) gene, which can also cause sudden cardiac death from ventricular arrhythmia. In particular, the NKX2-5 p.Arg25Cys missense variant has been repeatedly reported in patients with congenital heart defects and, more rarely, with hypogonadism. We report the case of a 7 year old boy with ventricular arrhythmias, thyroid dysgenesis and intellectual disability, born from consanguineous Tunisian parents. Exome sequencing and segregation analysis revealed two potentially relevant variants: the NKX2-5 p.Arg25Cys variant (maternally inherited), as well as a single heterozygous TSHR p.Gln90Pro variant (paternally inherited). Of note, a male sibling of the proband, presenting with intellectual disability only, carried the same two variants. No other TSHR variants, or other potentially relevant variants were identified. In this proband, despite the identification of variants in two genes potentially correlated to the phenotype, a definite genetic diagnosis could not be reached. This case report highlights the complexity of exome data interpretation, especially when dealing with families presenting complex phenotypes and variable expression of clinical traits.

INTRODUCTION

The human nucleotide kinase 2, homeobox 5 (NKX2-5) gene, located on chromosome 5q35.1 and encoding the homeobox-containing transcription factor NKX2.5, is known to be essential for normal heart morphogenesis, myogenesis and proper cardiac function (¹). Several inactivating mutations in the NKX2-5 gene, primarily located within the homeodomain, have been described in patients with a spectrum of congenital heart diseases (CHD), the most frequent ones being atrial or ventricular septal defects and tetralogy of Fallot (²). In addition, NKX2-5 pathogenic variants have been reported in adult patients with heart conduction defects such as atrial fibrillation (³) and, more recently, with sudden cardiac death from ventricular arrhythmia (⁴–⁶).

Besides its role in cardiac development, the presence of a fully functional NKX2-5 is essential for early thyroid morphogenesis, both for regulation of thyroidal cell differentiation and for proper cell migration to the final position in the neck (⁷). Accordingly, heterozygous NKX2-5 variants have also been linked to congenital hypothyroidism (CH) and thyroid dysgenesis (⁷–¹⁰).

It is well known that CH is associated with a significantly higher frequency of CHD (¹¹), as well as malformations of the urinary, gastrointestinal, and skeletal systems (¹²). While this can be attributed, in some cases, to the metabolic derangement due to the shortage of thyroid hormones, the impairment of genes involved in heart and thyroid organogenesis, such as NKX2-5, may also play a crucial role (⁷). Besides NKX2-5, several other genes have been involved in thyroid dysgenesis, such as the β-subunit of TSH (TSHB), the TSH receptor (TSHR), and another well-known transcription factor encoded by the paired-box gene 8 (PAX8) (¹³).

We report a patient with ventricular arrhythmias and thyroid dysgenesis, carrying two heterozygous variants in the NKX2-5 and TSHR genes. While variants in both genes are potentially related to these phenotypes, the interpretation of genetic results can be extremely complex, as illustrated by the patient described here.

CASE REPORT

Clinical features

A young boy, second-born to parents from Tunisia who are first grade cousins, was referred to our pediatric department. Born at 39 weeks of gestation after an uneventful pregnancy, his birth weight was 2250 g (-2.9 SDS), length 45 cm (-2.81 SDS), and head circumference 32.5 cm (-1.91 SDS).

CH was diagnosed at neonatal screening and confirmed at 6 days of life, with serum TSH 740 mU/L (reference range < 8.8 mU/L), FT4 0.9 pmol/L (13.9-26.1 pmol/L), and FT3 1.2 pmol/L (4.5-10.5 pmol/L). L-thyroxine treatment was promptly initiated with L-thyroxine 37.5 μg daily (15 μg/kg daily). At 6 months of life, a neck ultrasound (US) revealed thyroid hemiagenesis. During follow-up, L-thyroxine dose was adjusted for age and weight and thyroid function tests were always in the reference range. At 3 years of age, he displayed intellectual disability (ID) with a total IQ of 55 (WIPPSI III scale, normal values 85-115, deficit under 70), deep language impairment in production and understanding, and severe attention deficit.

In June 2019, at age 7 years and 3 months, he presented to the emergency room for malaise, nausea and vomiting, with episodes of loss of consciousness. ECG showed a large QRS tachycardia, right bundle branch block and left anterior hemiblock (Figure 1A). Vagal maneuvers and repeated adenosine i.v. (0.1 mg/kg and subsequently 0.2 mg/kg) were ineffective, therefore he underwent electrical cardioversion with success. Heart US was normal. Prophylaxis with low-dose verapamil 5 mg/kg daily was started during hospitalization and Holter ECG turned out negative. One month later, he presented another episode of ventricular fascicular tachycardia (Figure 1B); verapamil dosage was increased to 11 mg/kg daily and flecainide 60 mg/m² daily was started. Holter ECG showed very frequent ventricular ectopic beats (10% of the total), bimorphs isolated and in pairs, with a prevalent morphology; frequent ventricular beats to blanks, the longest of 26 beats and the fastest at a heart rate of 136 bpm.

Figure 1
On the ECG at 7 years and 3 month of age (Figure 1A), there was a large QRS tachycardia, right bundle branch block and left anterior hemiblock. One month later (Figure 1B), he presented again ventricular fascicular tachycardia.

On last examination, the patient was 9 years old; weight was -1.7 SDS and height -0.7 SDS. WISC IV psychometric test confirmed a moderate ID. He is on L-thyroxine 75 μg, verapamil 10 mg/kg and flecainide 60 mg/m² daily.

The mother suffers from acquired hypothyroidism (Hashimoto thyroiditis) diagnosed after the first pregnancy in September 2010. She is on levothyroxine 75 μg/day. Her ECG and cardiac ultrasound are normal. In the father, cognitive levels, ECG and thyroid function are normal. The propositus has one brother and two sisters: the boy is the first-born (January 2010) and displays ID (total IQ 45 at WISC IV scale), with severe deficit language disturbance in production and understanding, and normal array-CGH and FMR1 gene screening. Thyroid function and cardiac screening (cardiac US, ECG and Holter ECG) in the brother and the two sisters are normal (Figure 2).

Figure 2
Pedigree of the family harboring the c.73C>T mut. in NKX2.5 gene. The index patient is indicated by an arrow. Squares: male; circles: female; the transmission of the NKX2.5 variant is shown by a filled black area within each symbol, whereas the segregation of TSHR variant by a corresponding grey area. Results of thyroid and heart investigations are aligned with each symbol.

Genetic analysis

For WES, DNA libraries were enriched using Twist Human Core Exome Kit (Twist Bioscience), and sequenced on a NovasSeq6000 platform (Illumina, San Diego, CA). Bioinformatic analysis was performed as previously reported (¹⁴). Annotated variants were filtered and prioritized based on their quality, frequency in the population database gnomAD and predicted impact on the protein, with particular focus on genes implicated in CH and other thyroid defects. Identified variants were confirmed by Sanger sequencing. Accession numbers are NM_004387 for NKX2-5 and NM_000369 for TSHR.

WES analysis detected potentially relevant heterozygous variants in two genes (Figure 2):

NKX2-5 (nucleotide kinase 2, homeobox 5)

A recurrent missense variant Chr5(GRCh37):g.172662014G>A; c.73C>T, p.Arg25Cys, rs28936670 in NKX2-5 exon 1 was found in the proband, his brother affected with ID and their mother. According to American College of Medical Genetics and Genomics (ACMG) criteria, the variant is classified as a variant of uncertain significance (VUS), and has a minor allelic frequency of 0,343% in the gnomAD database. This variant has already been reported in several patients with CHD as well as healthy individuals of various ethnicities (¹⁵,¹⁶).

TSHR (TSH receptor)

The proband, his affected brother and two healthy sisters carried a heterozygous missense variant in TSHR Chr14(GRCh37):g.811534624_25AG>CT; c.269_270delinsCT; p.Gln90Pro inherited from their father. This variant is classified as likely pathogenic according to ACMG guidelines. No additional variants were detected in this gene.

DISCUSSION

Here we report the co-occurrence of thyroid hemiagenesis, ventricular arrhythmias and intellectual disability in a young patient. In subjects with CH, the incidence of extra-thyroidal malformations is reported to be fourfold higher than in control population, the commonest associated defects being CHD (¹¹,¹⁷). Whole exome sequencing and subsequent filtering detected potentially relevant heterozygous variants in two genes: NKX2-5 and TSHR. Yet, genetic results, despite the consanguinity of the parents, were not conclusive and did not allow to reach a definite diagnosis, as explained below.

Heterozygous variants in the NKX2-5 gene have been repeatedly reported in patients with CH and thyroid athyreosis, ectopy and hypoplasia, as well as with CHD (⁷–¹⁰). Mutations in this gene are estimated to account for approximately 4% of human cardiovascular malformations, most commonly secundum atrial septal defect (ASD), tetralogy of Fallot, and atrioventricular conduction disease (AVCD) (¹⁵,¹⁸–²⁰).

The identified p.Arg25Cys variant is a common variant often identified in patients with conotruncal heart anomalies (¹⁵,¹⁶), but to the best of our knowledge, it has never been described in young patients with ventricular arrhythmias (Table S1) (⁷,⁸,¹⁵,¹⁶,¹⁹–²⁵). Moreover, this variant was also reported in patients with thyroid ectopy, athyreosis and thyroid hypoplasia, but without cardiac malformations (⁷,⁸). Yet, the pathogenetic role of this variant is currently questioned, due to the high allelic frequency in specific populations (African-Americans) (¹⁵,¹⁶), as well as because of lack of segregation in families. Indeed, the incomplete penetrance and variable expressivity of this variant both at thyroidal and cardiac levels strongly suggest that other genes are expected to contribute to the phenotype.

Table S1
Previously reported patients and controls with the NKX2-5 p.Arg25Cys variant – Allelic frequencies are reported

References	Cardiac and/or thyroid anomaly		No. of patients carryng R25C	Family history	Other family members with R25C	No. Of control patients carryng R25C
Benson DW et al. (1999) (21)	TOF and VSD/no thyroid data		1/7	negative	not reported	0/50
Goldmuntz E et al. (2001) (15)	TOF/no thyroid data		3/114 (1,3%)	1 father: ASD	1 father	2/43 Africans Americans (2,3%)
McElhinney DB et al. (2003) (19)	7/608 (0,58%)no thyroid data	TOF Truncus arteriosus Interrupted aortic arch HLHS	1 1 1 1	father: VSD negative negative negative	not reported negative negative negative	0/50 randomcontrol Caucasian
Akcaboy MI et al. (2008) (16)	TOF/no thyroid data		1/72 (0,69%)	negative	father	2/185 Turkish (0,54%)
Stallmeyer B et al. (2010) (20)	HLHS/no thyroid data		1/121 (0,41%)	negative	negative	0/380 Caucasian
Perera JL et al. (2010) (4)	TOF/no thyroid data		1/159 (0,31%)	sister: VSD	brother	0/162 Brazilian
Rauch R et al. (2010) (22)	TOF/no thyroid data		2/230 (0,43%)	negative	negative	not tested
Beffagna G et al. (2013) (23)	2/100 (1%)	CoA, AVSD	1/100	negative	mother	0/250 Italian
		No thyroid defect
		ASD, VSD	1/100	negative	not tested
		Trisomy 21
		No thyroid defect
Dentice M et al. (2006) (7)	2/241 (0,41%)	Thyroid ectopy TSH 300 mU/L, FT4 5.4 pg/mL No cardiac malformation	1/241	negative	mother	1/561 (0,09%)
			Athyreosis	1/241	negative	father and brother
		TSH 419 mU/L, FT4 1.6 pg/mL
		No cardiac malformation
		Bilateral cortex atrophy
		Attention deficit hyperactivity disorder
Khatami M et al. (2017) (8)	2/65 (1,5%)	Thyroid hypoplasia TSH 17 mU/L, FT4 1.3 pg/mL No cardiac malformation	1/65	negative	negative	0/62 Iranian
	Thyroid hypoplasia TSH 21 mU/L, FT4 1.1 pg/mL No cardiac malformation		1/65	negative	negative
Pulignani S et al. (2018) (24)	TOF no thyroid data		1/17	negative	negative	not tested
Alcantara-Ortigoza MA et al. (2021) (25)	1	Trisomy 21 and normal heart	3/148	negative	mother	1/113 Mexican
	1	Trisomy 21 and perimembranous VSD and PDA			father
	1	Trisomy 21 and complete AVSD			mother negative, father not tested
In this study	1	Ventricular arrhythmias, no cardiac malformation Thyroid hemiagenesis. TSH 740 mU/L, FT4 0.9 pmol/L Intellectual disability	1	brother: intellectual disability	mother	not tested

• TOF: tetralogy of Fallot; VSD: ventricular septal defect; ASD: atrial septal defect; AVSD: atrioventricular septal defect; HLHS: hypoplastic left heart syndrome; CoA: aortic coartation. TSH normal range (0.35-4.5 mU/L), FT4 normal range (7.5-17.0 pg/mL, 12-22 pmol/L).

Functional studies on this variant also gave contrasting results. Arginine at codon 25 is conserved in mouse, rat and human, suggesting the potential deleterious consequences of a variation at this location. The residue lies just outside the first helix region of the functional domain; altering the charge from basic to neutral, the Cys25 variant may lead to a reduced hydrophobicity of the helix and the nearby region (¹⁶). Other functional studies showed for this variant a normal transcriptional activity and normal DNA binding to a monomeric site, but 3-fold reduction in DNA binding to dimeric sites, suggesting a slight impairment of dimerization (²), and normal DNA binding properties but impaired transactivation properties on target genes (⁷,²⁶).

In our family, the variant was found in the proband as well as in the brother, presenting only ID but no cardiac or thyroid defects, and in the mother, whose autoimmune thyroiditis is likely to be completely unrelated to this genetic finding.

Besides this, the proband also carried a single heterozygous variant (p.Gln90Pro) in the TSHR gene, present also in the other siblings and the healthy father. This variant is classified as likely pathogenic, and functional in vitro studies mutation showed 55% lower activity compared with the wild type protein (²⁷).

Recessive inactivating variants of the TSHR gene might manifest with a wide spectrum of thyroidal consequences: athyreosis at one end of the clinical range, mild hypothyroidism with normal thyroid gland size at the other far end (¹³). However, carriers of single heterozygous variants are healthy or present signs of subclinical hypothyroidism, i.e. hyperthyrotropinemia in euthyroidism (²⁸). Indeed, the identified variant had already been reported in two large pedigrees of Arabic descend, in which some members also carried a mutation in the TPO gene encoding thyroid peroxidase. In these families, those who were solely heterozygous carriers of the TSHR variant were either euthyroid, or showed a mild subclinical hypothyroidism or a very mild hypothyroidism, without any sign of thyroidal dysgenesis (²⁷).

While it is clear that none of the two variants is able to fully explain the thyroidal and cardiac manifestations of the proband, their potential contribution to the phenotype cannot be fully excluded and remains to be elucidated. Moreover, no relevant variants were identified that could potentially explain the intellectual disability in the patient and his similarly affected brother, as all variants surviving filtering in ID-related genes were all classified as variants of unknown significance and were inherited from one unaffected parent.

In conclusion, despite we acknowledge the great progress brought by large scale next generation sequencing to the diagnosis of complex genetic phenotypes, the patient reported here represents a paradigmatic example of how the interpretation of genetic variants can be an extremely difficult process, challenging genetic diagnosis and subsequent familial counselling.

• Data availability statement: the data that support the findings of this study are available on request from the corresponding author.
• Ethical statement: patient consents were obtained from all individuals from whom identifiable data are presented. This study was performed according to the Declaration of Helsinki protocol and approved by the local institutional review board.

REFERENCES

• ¹
  Weismann CG, Gelb BD. The genetics of congenital heart disease: a review of recent developments. Curr Opin Cardiol. 2007;22:200-6.
• ²
  Kasahara H, Lee B, Schott JJ, Benson DW, Seidman JG, Seidman CE, et al. Loss of function and inhibitory effects of human CSX/NKX2.5 homeoprotein mutations associated with congenital heart disease. J Clin Invest. 2000;106(2):299-308.
• ³
  Yu H, Xu JH, Song HM, Zhao L, Xu WJ, Wang J, et al. Mutational spectrum of the NKX2-5 gene in patients with lone atrial fibrillation. Int J Med Sci. 2014;11(6):554-63.
• ⁴
  Perera JL, Johnson NM, Judge DP, Crosson JE. Novel and highly lethal NKX2.5 missense mutation in a family with sudden death and ventricular arrhythmia. Pediatr Cardiol. 2014;35(7):1206-12.
• ⁵
  Jhaveri S, Aziz PF, Saarel E. Expanding the electrical phenotype of NKX2-5 mutations: Ventricular tachycardia, atrial fibrillation, and complete heart block within one family. Heart Rhythm Case Rep. 2018;4(11):530-3.
• ⁶
  Morlanes-Gracia P, Antoniutti G, Alvarez-Rubio J, Torres-Juan L, Heine-Suñer D, Ripoll-Vera T. Case Report: A Novel NKX2-5 Mutation in a Family With Congenital Heart Defects, Left Ventricular Non-compaction, Conduction Disease, and Sudden Cardiac Death. Front Cardiovasc Med. 2021;8:691203.
• ⁷
  Dentice M, Cordeddu V, Rosica A, Ferrara AM, Santarpia L, Salvatore D, et al. Missense mutation in the transcription factor NKX2-5: a novel molecular event in the pathogenesis of thyroid dysgenesis. J Clin Endocrinol Metab. 2006;91(4):1428-33.
• ⁸
  Khatami M, Heidari MM, Tabesh F, Ordooei M, Salehifar Z. Mutation analysis of the NKX2.5 gene in Iranian pediatric patients with congenital hypothyroidism. J Pediatr Endocrinol Metab. 2017;30(8):857-62.
• ⁹
  Cerqueira TL, Ramos Y, Strappa G, Martin DS, Jesus M, Gonzaga J, et al. The c.63A>G polymorphism in the NKX2.5 gene is associated with thyroid hypoplasia in children with thyroid dysgenesis. Arch Endocrinol Metab. 2015;59(6):562-7.
• ¹⁰
  Cerqueira TL, Ramos Y, Strappa G, Jesus M, Santos JG, Sousa C, et al. Mutation screening in the genes PAX-8, NKX2-5, TSH-R, HES-1 in cohort of 63 Brazilian children with thyroid dysgenesis. Arch Endocrinol Metab. 2018;62(4):466-71.
• ¹¹
  Olivieri A, Stazi MA, Mastroiacovo P, Fazzini C, Medda E, Spagnolo A, et al. A population-based study on the frequency of additional congenital malformations in infants with congenital hypothyroidism: data from the Italian Registry for Congenital Hypothyroidism (1991-1998). J Clin Endocrinol Metab. 2002;87(2):557-62.
• ¹²
  Reddy PA, Rajagopal G, Harinarayan CV, Vanaja V, Rajasekhar D, Suresh V, et al. High prevalence of associated birth defects in congenital hypothyroidism. Pediatr Endocrinol. 2010;2010:940980.
• ¹³
  Mio C, Grani G, Durante C, Damante G. Molecular defects in thyroid dysgenesis. Clin Genet. 2020;97(1):222-31.
• ¹⁴
  Borghesi A, Plumari M, Rossi E, Viganò C, Cerbo RM, Codazzi AC, et al. PUS3-related disorder: Report of a novel patient and delineation of the phenotypic spectrum. Am J Med Genet A. 2022;188(2):635-41.
• ¹⁵
  Goldmuntz E, Geiger E, Benson DW. NKX2.5 mutations in patients with tetralogy of Fallot. Circulation 2001;104(21):2565-8.
• ¹⁶
  Akçaboy MI, Cengiz FB, Inceoğlu B, Uçar T, Atalay S, Tutar E, et al. The effect of p.Arg25Cys alteration in NKX2-5 on conotruncal heart anomalies: mutation or polymorphism? Pediatr Cardiol. 2008;29(1):126-9.
• ¹⁷
  Kreisner E, Neto EC, Gross JL. High prevalence of extrathyroid malformations in a cohort of Brazilian patients with permanent primary congenital hypothyroidism. Thyroid. 2005;15(2):165-9.
• ¹⁸
  Benson DW. Genetic origins of pediatric heart disease. Pediatr Cardiol. 2010;31(3):422-9.
• ¹⁹
  McElhinney DB, Geiger E, Blinder J, Benson DW, Goldmuntz EJ. NKX2.5 mutations in patients with congenital heart disease. Am Coll Cardiol. 2003;42(9):1650-5.
• ²⁰
  Stallmeyer B, Fenge H, Nowak-Göttl U, Schulze-Bahr E. Mutational spectrum in the cardiac transcription factor gene NKX2.5 (CSX) associated with congenital heart disease. Clin Genet. 2010;78(6):533-40.
• ²¹
  Benson DW, Silberbach GM, Kavanaugh-McHugh A, Cottrill C, Zhang Y, Riggs S, et al. Mutations in the cardiac transcription factor NKX2.5 affect diverse cardiac developmental pathways. J Clin Invest. 1999;104(11):1567-73.
• ²²
  Rauch R, Hofbeck M, Zweier C, Koch A, Zink S, Trautmann U, et al. Comprehensive genotype-phenotype analysis in 230 patients with tetralogy of Fallot. J Med Genet. 2010;47(5):321-31.
• ²³
  Beffagna G, Cecchetto A, Dal Bianco L, Lorenzon A, Angelini A, Padalino M, et al. R25C mutation in the NKX2.5 gene in Italian patients affected with non-syndromic and syndromic congenital heart disease. J Cardiovasc Med (Hagerstown). 2013;14(8):582-6.
• ²⁴
  Pulignani S, Vecoli C, Borghini A, Foffa I, Ait-Alì L, Andreassi MG. Targeted Next-Generation Sequencing in Patients with Non-syndromic Congenital Heart Disease. Pediatr Cardiol. 2018;39(4):682-9.
• ²⁵
  Alcántara-Ortigoza MA, Sánchez-Verdiguel I, Fernández-Hernández L, Enríquez-Flores S, González-Núñez A, Hernández-Martínez NL, et al. Further Evidence That Defects in Main Thyroid Dysgenesis-Related Genes Are an Uncommon Etiology for Primary Congenital Hypothyroidism in Mexican Patients: Report of Rare Variants in FOXE1, NKX2-5 and TSHR. Children (Basel). 2021;8(6):457.
• ²⁶
  Esposito G, Grutter G, Drago F, Costa MW, De Santis A, Bosco G, et al. Molecular analysis of PRKAG2, LAMP2, and NKX2-5 genes in a cohort of 125 patients with accessory atrioventricular connection. Am J Med Genet A. 2009;149A(7):1574-7.
• ²⁷
  Sriphrapradang C, Tenenbaum-Rakover Y, Weiss M, Barkoff MS, Admoni O, Kawthar D, et al. The coexistence of a novel inactivating mutant thyrotropin receptor allele with two thyroid peroxidase mutations: a genotype-phenotype correlation. J Clin Endocrinol Metab. 2011;96(6):E1001-6.
• ²⁸
  Camilot M, Teofoli F, Gandini A, Franceschi R, Rapa A, Corrias A, et al. Thyrotropin receptor gene mutations and TSH resistance: variable expressivity in the heterozygotes. Clin Endocrinol (Oxf). 2005;63(2):146-51.

Publication Dates

• Publication in this collection
  13 Jan 2023
• Date of issue
  Feb 2023

History

• Received
  05 May 2022
• Accepted
  21 Aug 2022