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Journal of Electrocard

Hereditary long QT syndrome due to autoimmune hypoparathyroidism

in autoimmune polyendocrinopathy-candidiasis-ectodermal

dystrophy syndrome

Thomas Meyer, MD, PhD,a,b,4 Volker Ruppert, PhD,a

Konstantin Karatolios, MD,a Bernhard Maisch, MDa

aAbteilung Innere Medizin-Kardiologie, Philipps-Universitat Marburg, Baldingerstrasse, 35033 Marburg, GermanybAbteilung Psychosomatische Medizin und Psychotherapie, Philipps-Universitat Marburg, Baldingerstrasse, 35033 Marburg, Germany

Received 13 September 2006; accepted 14 December 2006

Abstract Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), also known as

0022-0736/$ – see fro

doi:10.1016/j.jelectroc

4 Corresponding

Philipps-Universit7t MTel.: +49 6421 28664

E-mail address: m

autoimmune polyglandular syndrome type I, is a rare autosomal recessively inherited disorder

characterized by variable combinations of endocrine and nonendocrine symptoms. In this report, we

describe two 20- and 17-year-old Turkish siblings presenting with typical symptoms of APECED,

including Addison disease, alopecia, vitiligo, and hypopituitarism, in whom electrocardiographic

examinations demonstrated an abnormal prolongation of the QT interval. In both cases, excessive

hypocalcemia due to primary hypoparathyroidism was identified as the underlying cause of the long

QT syndrome. Sequencing the gene coding for the autoimmune regulator revealed a homozygous

missense mutation in exon 14 with a C-to-T transition that resulted in the substitution of proline 539

for leucine in the carboxy-terminal protein molecule. Our data show that a single point mutation in

the transcriptional active autoimmune regulator protein is associated with inherited alterations in

calcium metabolism resulting from autoimmune reactions against the parathyroid glands. This

finding defines a congenital autoimmune disease as a hereditary long QT syndrome.

D 2007 Elsevier Inc. All rights reserved.

Keywords: Long QT syndrome; APECED; Autoimmune polyglandular syndrome type I; Hypoparathyroidism

Introduction

Autoimmune polyendocrinopathy-candidiasis-ectoder-

mal dystrophy (APECED), a genetic autoimmune disorder

inherited in a mendelian fashion, is characterized by an

immunologic breakdown of tolerance to self-antigens

resulting in organ-specific autoimmune reactions toward

different endocrine and nonendocrine tissues. The clinical

phenotype of the disease is highly variable and includes the

failure of multiple endocrine glands, such as the adrenal

cortex, the gonads, the thyroid and parathyroid glands, and

the pancreatic b cells.1-6 The diagnosis is based on the

occurrence of 2 of the 3 major clinical manifestations, that

is, primary atrophic hypoparathyroidism, primary adreno-

cortical failure (Addison disease), and chronic mucocutane-

nt matter D 2007 Elsevier Inc. All rights reserved.

ard.2006.12.013

author. Klinik fqr Innere Medizin-Kardiologie

arburg, Baldingerstrasse, 35033 Marburg, Germany

62; fax: +49 6421 2868954.

eyert@med.uni-marburg.de

,

.

ous candidiasis. APECED also frequently leads to charac-

teristic ectodermal symptoms (pitted nail dystrophy, dental

enamel hypoplasia, alopecia, and vitiligo) and may be

associated with gastrointestinal manifestations, such as

malabsorption, autoimmune hepatitis, and chronic atrophic

gastritis with pernicious anemia.2,4-8 The clinical presenta-

tion of APECED is very heterogeneous in terms of age at

onset, number of components, and time span between

development of new symptoms.1 The clinical manifestations

most likely result from destruction of target organs by cell-

and antibody-mediated attack. One of the features indicating

the autoimmune nature of APECED is the presence of

lymphocytic infiltrations in the affected tissues. Several

autoantibodies recognizing cytochromes involved in the

biosynthesis of steroid hormones or enzymes catalyzing

steps in neurotransmitter synthesis have been detected in the

serum of APECED patients, although their precise role in

the pathogenesis of APECED is unknown.4,6,9-13

The gene defective in APECED was identified by

positional cloning on chromosome 21 (21q22.3) and

iology 40 (2007) 504–509

Fig. 2. Family pedigree showing the identified genotypes found in each

subject. Filled circles and squares represent affected female and male

subjects, respectively. A half-filled square represents a male subject with a

mild form of APECED; and open circles and squares represent unaffected

female and male subjects, respectively.

T. Meyer et al. / Journal of Electrocardiology 40 (2007) 504–509 505

encodes a nuclear protein consisting of 545 amino acids,

termed autoimmune regulator (AIRE).14 The domain

structure of AIRE has several features of typical transcrip-

tional regulators; and indeed, the protein functions as a

powerful transcriptional transactivator in vitro when fused

to a heterologous DNA binding domain.2,15 Furthermore,

AIRE has been shown to interact with the common

transcriptional co-regulator CREB-binding protein, adding

further strength to the hypothesis that this protein is

involved in transcriptional control.15 The expression of

AIRE is restricted mainly to tissues having an important role

in the maturation of the immune system, such as thymus,

lymph node, spleen, and fetal liver. In the thymus, AIRE is

expressed in 2 types of antigen-presenting cells, namely,

medullary thymic epithelial cells and thymus monocyte-

derived dendritic cells.6,16,17 Autoimmune regulator pro-

motes self-tolerance by inducing the expression of a battery

of peripheral-tissue antigens in these cells. The increased

antigen-presentation capability of thymic stromal cells

induces the tolerization of thymocytes by negative selection

of self-reactive T cells.18-20

Whereas malfunctions in the diverse endocrine pathways

are the diagnostic hallmark of the disease, much less

attention has been paid on cardiac symptoms. In this report,

Fig. 1. Clinical presentation of a 20-year-old patient with APECED

syndrome showing oral candidiasis (A, indicated with arrows) and

ectodermal symptoms such as nail dystrophy, vitiligo, and alopecia (B).

we describe 2 siblings presenting with APECED syndrome

who both developed a potentially harmful symptom,

namely, marked prolongation of the QT interval as a

consequence of hypocalcemia-induced hypoparathyroidism.

These findings suggest that the autoimmune polyglandular

syndrome type I has to be added to the list of hereditary long

QT syndromes.

Case reports

Patient 1

A 20-year-old man with APECED was referred to the

cardiological department because of a lengthening of the QT

interval seen in an electrocardiogram (ECG) recording at

admission. The patient of consanguineous, healthy parents

was born in Turkey. In the first year of his life, the patient

had recurrent bronchial infections. He developed mycotic

paronychia by Trichophyton rubrum in early infancy that

was treated unsuccessfully with griseofulvine. Patchy hair

loss was first visible at the age of 2 years and since then

progressed continuously to alopecia universalis. Before the

diagnosis of Addison disease at the age of 4 years, the

patient had pneumonia. Hypoglycemia, fatigue, and dehy-

dration associated with low sodium and chloride serum

levels were suggestive of adrenocortical failure. The

diagnosis was confirmed by the finding of reduced serum

cortisol and aldosterone concentrations in combination with

an elevated corticotropin level. Thus, oral substitution with

hydrocortisone and fludrocortisone was started. In the same

year, the serum calcium and parathormone levels were

found to be reduced. Bone densitometric measurements

suggested osteoporosis, and a medication with vitamin D

commenced. Recurrent urolithiasis and nephrocalcinosis

due to hypercalciuria were reported. Later, pernicious

anemia manifested; and autoantibodies against intrinsic

factor were detected. Immunofluorescence tests revealed

autoantibodies against adrenocortical tissue. As additional

symptoms of the underlying autoimmune disease, the

patient developed vitiligo and chronic mucocutaneous

candidiasis (Fig. 1A). Pitted nail dystrophy and dental

enamel hypoplasia were interpreted as signs of ectodermal

dystrophy (Fig. 1B). According to his medical records,

pubertas tarda was diagnosed and treated intermittently with

Fig. 3. Twelve-lead ECG recording taken from patient 1 demonstrating sinus rhythm and a significant prolongation of the QT interval.

T. Meyer et al. / Journal of Electrocardiology 40 (2007) 504–509506

subcutaneous administration of recombinant somatropin.

After successfully leaving grammar school, the patient was

readmitted to the hospital because he progressively had

fatigue and asthenia. In the clinical examination, a positive

Chvostek test response and signs of tetany could be elicited.

Blood pressure (100/70 mm Hg) and heart rate (HR;

64/min) were normal. Serum concentrations of total and ion-

ized calcium were markedly reduced (1.3 and 0.8 mmol/L,

respectively). In contrast, the serum phosphate concentra-

tion was elevated (2.1 mmol/L). The levels of parathyroid

hormone (0.3 ng/L; reference range, 11-65 ng/L) and

cortisol (40 lg/L; reference range, 45-220 lg/L) were

reduced. Potassium and magnesium concentrations (3.6 and

0.7 mmol/L, respectively) were normal. Serum concentra-

tions of testosterone (0.3 lg/L), follicle-stimulating hor-

mone (12 IU/L), and luteinizing hormone (21 IU/L) were

T. Meyer et al. / Journal of Electrocardiology 40 (2007) 504–509 507

suggestive of hypergonadotrophic hypogonadism. Prolactin

level was within the reference range. Thyroid function was

normal (thyrotropin, 4.3 mU/L; free T4, 11 pmol/L; free T3,

5.3 pmol/L) in the presence of antithyroid peroxidase

(86 IU/mL; reference range, b40 IU/mL) and antithyroglo-

bulin (145 IU/mL; reference range, b60 IU/mL) antibodies.

Patient 2

The reported patient 1 has an older brother and 2 sisters

(Fig. 2). The 27-year-old brother had developed nail

dystrophy and a mild form of alopecia areata, but no signs

of endocrinopathy. A 24-year-old sister is healthy; and a

younger sister, who is now 17 years old, also had typical

symptoms of APECED. The affected sibling developed nail

dystrophy early in her life. In early infancy, alopecia areata

was observed, which progressed discontinuously. In addi-

tion, the patient noticed a focal depigmentation of the skin,

particularly in the face and hands. At 3 years of age,

recurrent episodes of generalized seizures occurred while

the serum calcium concentration (1.8 mmol/L) was mark-

edly decreased. Hypercalciuria was noticed. The low serum

parathormone concentration (5 ng/L) detected at that time

suggested the onset of autoimmune hypoparathyroidism.

Since that time, the patient was set on an oral substitution

with calcium in combination with calcitriol. When she was

7 years old, she complained about nonsuppurative otitis

media with tympanic membrane perforation. At 9 years of

age, serum cortisol levels were low despite elevated

corticotropin levels and failed to increase after corticotropin

stimulation. Autoantibodies against adrenal tissue became

positive. Morbus Addison was diagnosed and treated with

hydrocortisone and fludrocortisone. As a further manifesta-

tion of polyendocrinopathy, she developed autoimmune

thyroiditis requiring a substitution with thyroxine. At the

age of 14 years when she presented with ketoacidotic coma,

diabetes mellitus was diagnosed. Human insulin was

administered, but the management of diabetes mellitus

was complicated. Because of recurrent episodes of symp-

tomatic hypo- and hyperglycemia (HbA1c, 12.4%), the

diabetes was ultimately treated with an external insulin

pump. Hypopituitarism that manifested with a retarded

growth and pubertal development was diagnosed, and

treatment with somatropin was started. Oral candidiasis

was diagnosed. In repeated measurements, the serum

concentrations of ionic and total calcium (0.8 and

1.3 mmol/L) remained below the reference range despite

oral administration of calcium and calcitriol. Serum levels of

potassium (4.1 mmol/L) and magnesium (0.7 mmol/L)

were normal.

ig. 4. Electropherograms showing a single base exchange in exon 14 at

ucleotide position 1743 in the AIRE gene. In patient 1, DNA sequencing

vealed a point mutation with a C-to-T transition (mutant base underlined)

at resulted in a substitution of proline 539 for leucine in the carboxy-

rminal region of the AIRE protein (A). A sibling of patient 1 was

eterozygous at this nucleotide position (B), and a normal control subject

showed the wild-type sequence (C).

ECG findings

A standard automatic 12-lead ECG was obtained at a

paper speed of 50 mm/s. For analysis of HR-corrected QT

intervals (QTc), the HR based on individual R-R intervals

(ie, intervals between 2 consecutive R waves) and QT

intervals were measured in the chest lead with maximal

T-wave amplitude. The QT intervals were measured from

the first deflection of the QRS onset to the end of the Twave

as it merged with the isoelectric baseline. For correction of

QT intervals, the following QTc formulae were used: Bazett

(QTcB = QT [HR / 60]1/2), Fridericia (QTcFri = QT [HR /

60]1/3), Framingham (QTcFr = QT + 154 [1 � 60 / HR]),

and Hodges (QTcH = QT + 1.75 [HR � 60]).21 QTc

intervals were averaged over 5 consecutive cardiac beats

and presented in milliseconds.

In patient 1, the ECG obtained at admission was

unremarkable, except for a significantly prolonged QT

interval (QTcB, 533 milliseconds; QTcFri, 499 millisec-

onds; QTcFr, 488 milliseconds; and QTcH, 489 milli-

seconds, respectively) (Fig. 3). The lengthening of the QT

interval was seen also in consecutive ECG recording. After

oral substitution of calcium, the prolonged QTc values

reached the cutoff level reported for normal ECGs (QTcB,

486 milliseconds; QTcFri, 465 milliseconds; QTcFr,

462 milliseconds; and QTcH, 462 milliseconds). Similarly,

in patient 2, the ECG revealed a normofrequent sinus

rhythm with marked prolongation of the rate-corrected QT

intervals (QTcB, 529 milliseconds; QTcFri, 511 milli-

seconds; QTcFr, 505 milliseconds; and QTcH, 500 milli-

seconds). Repeated Holter recordings demonstrated

decreased HR variability, but no episodes of ventricular

tachycardia or torsade de pointes.

Identification of AIRE gene mutation

After written informed consent was obtained, blood

samples were taken from all family members. Genomic

DNA was extracted from isolated peripheral blood mono-

nuclear cells. The exons of the AIRE-1 gene were then

polymerase chain reaction (PCR)–amplified using 14 sets of

intronic primer pairs as described by Wang and col-

leagues.22 The PCRs were carried out in a final volume of

25 lL containing 0.3 lmol/L of each primer, 25 ng genomic

F

n

re

th

te

h

Fig. 5. Mutational screening of family members for the P539L mutation in

the AIRE gene as determined byMspI enzymatic digestion. The mutation at

nucleotide position 1743 of the AIRE cDNA abolishes an MspI restriction

site (5V-CCGG-3V). In both APECED patients, an 86-bp product was

detected, but no 52-bp fragment, indicating that they are homozygous. Both

parents and a brother were heterozygous as judged by the detection of all 3

relevant fragments. In the healthy sister and an unrelated control subject, 2

wild-type alleles were identified. Lane 1: father (F), lane 2: mother (M),

lane 3: patient 1 (P1), lane 4: patient 2 (P2), lane 5: brother (B) with mild

symptoms, lane: 6 healthy sister (S), and lane 7: unrelated control

subject (C).

T. Meyer et al. / Journal of Electrocardiology 40 (2007) 504–509508

DNA, and 1 U Taq DNA polymerase. The PCR consisted of

35 cycles of 30 seconds at 948C for denaturing, 30 seconds

at optimal annealing temperature (588C-628C), and 30 sec-

onds at 728C for extension, preceded by 7 minutes at 728C.After PCR amplification, the products were electrophoresed

on a 1.5% agarose gel and purified by affinity chromatog-

raphy. The DNA sequencing was performed using an

Applied Biosystems 310 automated sequencer.

In patient 1, a point mutation was identified in exon 14 of

the coding sequence of the AIRE-1 gene (Fig. 4A). At

nucleotide position 1743, a C-to-T transition resulted in

a change of a single amino acid residue. The gene codes for

a mutant protein with a substitution of proline 539 for

leucine in the carboxy-terminal protein molecule. In both

parents and the oldest sibling, DNA sequencing revealed

heterozygosity at this nucleotide position (Fig. 4B), where-

as the sequence was wild-type in the unaffected sister

and a healthy control subject (Fig. 4C). In addition to

this missense mutation, a single nucleotide polymorphism

was found in intron 9 (11107 G to A, not shown) of the

AIRE gene.

The presence of the P539L mutation was confirmed by

MspI enzymatic digestion (Fig. 5). The mutation abolishes

an MspI restriction site (5V-CCGG-3V) in exon 14 so that,

instead of two 34– and 52–base pair (bp) fragments, an

uncleaved 86-bp product occurs. In patients 1 and 2, only

the high molecular product was detectable, but not the

cleaved 52-bp fragment. Both parents and a brother proved

to be heterozygous because all 3 restriction fragments were

detected (34, 52, and 86 bp). In the healthy sister and an

unrelated control subject, 2 wild-type alleles were identified

(34 and 52 bp).

Discussion

In this contribution, we describe the occurrence of an

inherited long QT syndrome due to excessive hypocalcemia

in 2 siblings with autoimmune polyglandular syndrome

type I. Both patients fulfill the classic criteria for diagnosing

APECED because they developed the classic triad of

polyendocrinopathy, oral candidiasis, and ectodermal dys-

trophy already in early childhood. Molecular analysis

revealed homozygosity for a point mutation in exon 14 of

the AIRE gene that coded for a mutant protein with a

nonconservative amino acid exchange in the carboxy-

terminal molecule region. The mutation resulted in a

substitution of proline to leucine in position 539 at the

carboxy terminus of the AIRE protein. The same P539L

mutation had been described before by Meloni and

colleagues in a patient from southern Italy presenting with

typical APECED.5

Restriction length polymorphism confirmed that the 2

affected siblings were homozygous, whereas the parents

were heterozygous. Another sister apparently free of

symptoms had 2 wild-type alleles, and an older brother

was found to be heterozygous. The latter one presented with

mild features of APECED including alopecia areata and nail

dystrophy, but no signs of endocrinopathy. The occurrence

of an incomplete clinical presentation of the syndrome in a

patient with a characteristic mutation in heterozygosity

poses into question whether APECED is correctly classified

as a strict autosomal-recessive disorder.8 Rather, the finding

of more than one typical clinical features of APECED in our

heterozygous patient argues for a refinement of the

diagnostic criteria used to define the syndrome.

The abnormally low serum calcium levels in our patients

seen in repeated measurements resulted from atrophic

hypoparathyroidism, which is a cardinal symptom of

APECED caused by the infiltration and subsequent destruc-

tion of the parathyroid glands by autoreactive T cells. No

other cause of QT prolongation was found; and in particular,

a pharmacologically induced prolongation of the QT

interval could be excluded, which accounts for most of

the acquired long QT syndromes. Moreover, calcium

substitution led to a shortening of the QT interval, thus

indicating that indeed the low calcium concentration is the

pathophysiological basis of the ECG abnormalities.

Compared with the other causes of long QT syndromes,

hypocalcemia-induced QT interval prolongation seems to

be rare. In patients with abnormally low serum calcium

levels, iatrogenic causes such as aggressive diuretic

treatment, hemodialysis in end-stage renal insufficiency,

and complete hypoparathyroidectomy have been described

that account for most of the cases with prolonged QT

intervals due to hypocalcemia.23-25 It is well known that

abnormalities in calcium metabolism alter the repolarization

phase of the myocardium. Inward Ca2+ currents are one of

the factors determining the plateau configuration of the

action potential in cardiomyocytes, and hypocalcemia

prolongs phase 2 of the action potential and thus prolongs

the repolarization time. As seen in our case, significant

prolongation of the QT interval due to hypocalcemia may

not be associated with inversion or other morphological

alterations of the T wave.

Recently, Buzi and coauthors reported on a 7-year-old

patient presenting with minor facial dysmorphism, mild

mental retardation, and undetectable parathyroid hormone

levels, but no further clinical features of APECED, in which

a typical AIRE mutation (R257X) was detected on a single

allele only.8 This patient had a prolonged QT interval;

however, details of the ECG recordings and, in particular,

T. Meyer et al. / Journal of Electrocardiology 40 (2007) 504–509 509

the lengthening of the QT interval were not presented. The

occurrence of an incomplete clinical presentation of

APECED together with a typical AIRE mutation in

heterozygosity found in this and 2 other patients described

does not fit into the classic definition of the syndrome and

poses questions about whether these patients should be

considered affected by this condition.8

Our findings suggest that APECED may be added to the

list of hereditary long QT syndromes. Whereas all inherited

long QT syndromes genetically elucidated so far are caused

by defective mutations in genes coding for different cardiac

ion channels, the pathophysiology of APECED involves

severe electrolyte imbalances due to congenital autoimmune

reactions against hormone-producing tissue. Structural

changes in cardiac ion channels as well as genetically

inherited alterations in calcium homeostasis both lead to

abnormal ventricular repolarization. The hypoparathyroid-

ism-induced hypocalcemia found as a typical symptom in

APECED patients should focus our attention to concomitant

ECG abnormalities, particularly the prolongation of the QT

interval, to assess the incidence of potentially malignant

polymorphic ventricular tachycardia in this inherited auto-

immune syndrome.

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