decrease in thyrocalcitonin-containing cells and analysis of other congenital anomalies in 11...

American Journal of Medical Genetics 46641-646 (1993)

Decrease in Thyrocalcitonin-Containing Cells and Analysis of Other Congenital Anomalies in 11 Patients With DiGeorge Anomaly Jose Palacios, Carlos Gamallo, Marcia1 Garcia, and Jose Ignacio Rodriguez Department of Pathology, La Paz Hospital, Madrid, Spain

Experimental studies have demonstrated that the DiGeorge anomaly is due to cranial neural crest abnormalities. In the present study, the quantitation of thyrocalcitonin immunoreactive cells (C-cells) has been used to evaluate whether cells derived from the cranial neural crest are or are not present in normal propor- tions in patients with this anomaly. Thyroid sections from 11 such patients and 11 control patients were studied immunohistochemi- cally at autopsy in order to determine the number and distribution of thyrocalcitonin- containing cells. Patients with DiGeorge anomaly showed thymic and parathyroid aplasia/hypoplasia and cardiovascular defects, such as type B interrupted aortic arch, truncus arteriosus and tetralogy of Fallot. Other associated defects were alobar holoprosencephaly and meningocele (previously unreported defects in this anomaly), arhinencephaly, renal cystic dysplasia, ureterohydro- nephrosis, esophageal atresia with tra- cheoesophageal fistula, and cleft lip and palate. The volume density of C-cells (1.187%) and the mean number of C-cells per follicle (1.42) was significantly lower in patients with DiGeorge anomaly than in control patients (3.475% and 2.367, respectively). These results indicate a decrease in cells derived from the neural crest in patients with DiGeorge anomaly, and support the hypothesis of a neural crest disturbance as the pathogenetic factor responsible for this anomaly.

KEY WORDS: neural crest, thyrocalcitonin, holoprosencephaly, CNS anomalies

6 1993 WfieY-bS, hC.

Received for publication September 28, 1992; revision received February 23, 1993.

Address reprint request to Dr. Jose Ignacio Rodriguez, Departa- mento de Anatomia Patologica, Hospital La Paz, Paseo de la Cas- tellana 261, E-28046 Madrid, Spain.

0 1993 Wiley-Liss, Inc.

INTRODUCTION The association of an absence of thymus and para-

thyroid, together with craniofacial and cardiac malformations, has classically been known as DiGeorge syndrome. However, the observation of causal heterogeneity among patients with this characteristic malformative pattern, and the hypothesis that these anomalies originate in abnormalities of the cephalic neural crest cells, lead Lammer and Opitz [1986] to suggest that DiGeorge “syndrome” is actually a polytopic devel- opmental field defect (DiGeorge anomaly).

With respect to the pathogenesis of DiGeorge anomaly, Robinson [19751 proposed that it was due to a defi- cient blood supply of the third, fourth, and fifth embry- onic pharyngeal pouches secondary to a premature involution of the thyroidea ima artery. More recently, Bockman and Kirby [19841 showed that ablation of pre- migratory cephalic neural crest cells in chick embryos produced the DiGeorge anomaly phenotype, including thymic aplasia or hypoplasia and aorticopulmonary septation abnormalities. These authors suggested that DiGeorge anomaly probably was related to failure of neural crest cells to migrate and interact in sufficient quantity so as to support the development of the organs in question. Other experimental studies using teratogens (i.e., alcohol, retinoids) also support this hypothesis [Sulik et al., 1986; Webster et al., 19861. The cephalic neural crest provides all the mesenchymal cells in the facial skeleton and pharyngeal arch derivatives. Kirby and Waldo [ 19901 have proposed the term “cardiac neural crest” to designate the region of the cranial neural crest between the midotic placode and the caudal limit of somite 3. This region provides cells which form the walls of the aortic arch arteries (excluding endo- thelium), the connective tissue of the thymus and para- thyroids, and which also migrate into the cardiac out- flow tract. However, the only paper to demonstrate a reduction of cells derived from the neural crest in human patients with the DiGeorge anomaly is the one by Burke et al. [19871. These authors reported a decreased number of C-cells (parafollicular cells) in the thyroid gland of affected infants. To the best of our knowledge no further studies have confirmed these findings.

The aim of this report is to confirm DiGeorge anomaly, irrespective of its primary cause, as a neurocristopathy.

642 Palacios et al.

The present quantitative analysis in patients with the DiGeorge anomaly has documented a significant reduction in thyroid C-cell numbers, confirming the Burke et al. [19871 findings, and supporting the hypothesis of a neural crest defect as the pathogenetic factor for Di- George anomaly. In addition, 3 patients in this series had associated central nervous system anomalies previously unreported in the DiGeorge anomaly.

MATERIALS AND METHODS Among our pediatric autopsies files there were 11

patients with DiGeorge anomaly in which thyroid gland samples through one or both lateral lobes at the upper two-thirds level were available. All patients were the product of uncomplicated term pregnancies in which neither teratogenic exposures nor a familial history of malformations were recorded. Patients were 8 males and 3 females, ranging from 1 day to 1 year of postnatal age, and from 2,500 to 8,000 g ofbody weight. Eleven infants, matched for the study patient's weight and extrauterine age, were used as control patients. Control patients had no malformations and their main pathological processes at autopsy were pneumonia and/or meningococceal sepsis.

Tissue samples were fixed in 10% buffered formalin. A transverse slice from the upper two-thirds of the thyroid gland lobes was processed for paraffin embedding. Sec- tions were cut a t 4 pm, and stained with haematoxylin and eosin. For the calcitonin light microscopic immu- nohistochemical study sections of paraffin-embedded tissue were cut a t a thickness of 3 Frn and de- paraffinized. After rehydration, the tissue sections were pretreated with hydrogen peroxide in methanol to elim- inate endogenous peroxidase activity. The sections were incubated with diluted normal goat serum to block non- specific antibody reactions, and incubated for 2 hours at 37°C with rabbit anti-calcitonin antibody (prediluted) (Lipsaw). For the control study, diluted normal rabbit serum was used as the first antibody. The sections were then treated with biotinylated goat anti-rabbit antibody (dilution 1:400) (Biomakor). After washing with Tris buffer, the sections were treated with extravidin-peroxidase (dilution 1:800) (Biomakor) and washed with "ris buffer. Finally, the sections were developed with dia- minobenzidine-hydrogen peroxide solution and counter- stained with Mayer's hematoxylin.

C-cell volume density (relation of volume of C-cells and thyroid volume) in thyroid sections was measured by the direct projection of microscopic slide images on a 168 point matrix [Weibel, 19791. The observer was un- aware of any data concerning the patient in order to avoid the eventual bias of examining areas with poor C-cell content. We also calculated the mean number of these cells per follicle (considering only those follicles with at least one immunoreactive cell).

One Mann-Whitney U was used for statistical analysis of the data.

RESULTS The most relevant clinical data and pathological find-

ings are summarized in Table I. Heart failure and/or

cyanosis were observed clinically in 7 patients. Five patients who survived more than one month died of infectious disease and sepsis; 4 of these also developed hypocalcemia. A clinical diagnosis of DiGeorge anomaly was made in only 3 patients (patients 2,5, and 7), based on immunological and/or biochemical studies. Minor facial anomalies, such as hypertelorism and apparently low-set ears, were recorded in some patients (Fig. 1). Patient 11 showed the facial appearance of the premax- illary agenesis type of holoprosencephaly (Fig. 2). Chro- mosomal analysis were performed in only two patients (5 and 11). Both patients had an apparently normal 46,XY karyotype. Since some anomalies associated with syndromes which are the primary cause of DiGeorge anomaly cannot be detected in infancy or autopsy (mental retardation, behavioral anomalies, and even many structural anomalies) no effort was made to reach a primary diagnosis.

The autopsy documented the absence of parathyroid glands in all patients and thymus agenesis in 9 patients. Two infants had an asymmetric hypoplastic thymus. Ventricular septa1 defect was present in all but 2 patients, type B interrupted aortic arch in 3, truncus arteriosus in 2, and Fallot's tetralogy in one. Other associated congenital malformations, mainly affecting the central nervous and genitourinary systems, were found in 5 patients.

The comparison between thyroid C-cell data from patients and control infants is shown in Table 11. The percentage of cases with at least one C-cell in the whole thyroid gland section was 80% in DiGeorge patients, and 100% in the control group. A statistically significant decrease of C-cell volume density and C-cell number per follicle was found in the patient group.

Fig. 1. Facial appearance of patient 9.

TAB

LE I

. M

ain

Man

ifes

tatio

ns a

nd P

atho

logi

cal

Find

ings

in

11 D

iGeo

rge

Patie

nts*

case

se

x 1

M

2 F

3 M

4 M

5 M

6 F

7 M

8 M

9

M

10

F

11

M

Wei

ght

Age

at

at d

eath

2 m

o 3,

220

deat

h (g

)

2 m

o 2,

660

2d

3,

050

5 m

o 6,

500

2 m

o 2,

500

Id

1,

900

1 yr

8,000

7d

2,

540

3d

2,

900

1 m

o 2,

660

Id

2,

900

Pres

entin

g co

mpl

aint

In

trac

tabl

e di

arrh

ea

Cya

nosi

s

Hea

rt fa

ilur

e

Hyp

ocal

cem

ia

Lum

bar

men

ingo

cele

H

ydro

ceph

aly

Evol

utio

n C

andi

da a

nd C

MV

in

fect

ion

Hyp

ocal

cem

ia

seps

is

Seps

is

Hyp

ocal

cem

ia

seps

is

Thy

mus

Pa

rath

yroi

d C

ardi

ac d

efec

t A

A

N

o

A

A

TA; V

SD

A

A

VSD

; ASD

;

H

A

No

A

A

VSD

IAA

A

A

VSD

C ya

nosi

s H

ypoc

alce

mia

A

A

Fa

llot

Hea

rt f

ailu

re

A

A

VSD

; MA

H

eart

fai

lure

A

A

V

SD;

IAA

; C

ystic

kid

ney

unic

uspi

d A

V

Hea

rt f

ailu

re

left

lob

e A

do

uble

AA

;

MC

A;

A

A

VSD

; TA

; ASD

seps

is c

hick

enpo

x

agen

esis

H

LV; A

SD

cvan

osis

Ass

ocia

ted

defe

ct

Non

e

Non

e

righ

t meg

aure

ter

Non

e L

umba

r m

enin

goce

le

Alo

bar

holo

pros

ence

phal

y; M

ecke

l di

vert

icul

um; m

obile

cec

um;

hypo

plas

tic u

teru

s; a

tret

ic ce

rvix

N

one

Non

e D

yspl

astic

rig

ht k

idne

y

Non

e

Arh

inen

ceph

aly;

PA

; eye

col

obom

a;

left

ure

tero

hydr

onep

hros

is

AE

/FT

E

* A: a

gene

sis;

H: h

ypop

lasi

a; T

A: t

runc

us ar

teri

osus

; VSD

: ven

tric

ular

sept

al de

fect

; ASD

: aur

icul

ar se

ptal

def

ect;

HLV

: hyp

opla

stic

left

ven

tric

le; I

AA

: int

erru

pted

aorti

c ar

ch;

AV

: aor

tic v

alve

; MC

A: m

ultip

le c

onge

nita

l ano

mal

ies;

PA

: pre

max

illar

y ag

enes

is.

644 Palacios et al.

Fig. 2. F'remaxillary agenesis in patient 11.

DISCUSSION These DiGeorge patients also showed the variable

pathological picture reported in this polytopic field defect [Conley et al., 19791. The typical findings in this anomaly are absence or hypoplasia of the thymus and parathyroid glands, which are, respectively, responsible for defective cellular immunity and hypocalcemia [Di- George, 19651. Cardiovascular malformations are present in 90% of the patients, and are the major cause of death [Moerman et al., 19801. The most frequent cardiac malformations are aortic arch and conotruncal defects, mostly type B interrupted aortic arch and persistent truncus arteriosus [Conley et al., 1979; Moerman et al., 1980; Marmon et al., 1984; Radford et al., 1988; Van Mierop and Kutsche, 19861. In addition, patients with DiGeorge anomaly frequently have minor facial anomalies characterized by hypertelorism, apparently low-set

ears, short philtrum, and retrognathia [Robinson, 19751.

Several other congenital malformations have been documented in this anomaly. Conley et al. [19791 reported that, as in our series, malformations of the renal and central nervous systems are the most frequent system defects associated with DiGeorge anomaly. These authors reported CNS malformations in 6 of their 25 patients (24%) including arhinencephaly (3 patients), lissencephaly (1 patient), simple gyral pattern (1 patient), and microcephaly (1 patient). Although no one else has reported a similar incidence of CNS malformations, the rate of CNS defects in our series is similar to that reported by Conley et al. [19791.

After the Conely et al. [19791 report DiGeorge anomaly was included among the conditions in which anomalies of the holoprocencephalic spectrum may occur [Cohen, 1982,19891. However, to the best of our knowledge, only arhinencephaly (isolated or associated with Kallmann syndrome) has been documented [Conley et al., 1979; Shen et al., 19791. Alobar holoprosencephaly, as in our patient 6, has not been reported previously in DiGeorge anomaly. Thymic anomalies, such as hypoplasia, unilateral hypoplasia, partial agenesis, and asymmetry may occur in holoprosencephalic infants [Siebert et al., 19901.

CNS malformations such as those observed in Di- George anomaly have been reported in the CHARGE association [Lin et al., 19901, a condition which not only shares manifestations with DiGeorge anomaly, but may also be found in these patients [Dische, 1968; Barret et al., 1981;'Pagon et al., 1981; Siebert et al., 19851. Holo- prosencephaly has been found associated with velo- cardio-facial syndrome [Wraith et al., 19851, which is a known cause of DiGeorge anomaly.

No cases of neural tube defects have been reported previously in DiGeorge anomaly series, and in anen- cephaly/spina bifida series no cases of DiGeorge anomaly have been observed. Thus, our patient 5 seems to be the first reported association between DiGeorge anomaly and neural tube defect.

Hydronephrosis, renal microcysts, and renal cystic

TABLE 11. Data Obtained From Patients With DiGeorge Anomaly (DG) Compared With Those From a Control Group (C) C-cells volume

Postnatal age (d) Body weight (g) C-cells No./follicle density (%)

No. C DG C DG C DG C DG 1 52 65 4,500 3,200 3.42 2.00 4.76 0.70 2 50 60 2,500 2,660 2.14 2.12 2.67 1.98 3 2 2 2,200 3,050 2.00 1.25 1.19 1.50 4 60 150 5,000 6,500 2.57 1.62 7.34 0.70 5 75 60 3,350 2,850 2.37 1.37 4.80 1.23 6 3 2 650 1,990 2.00 0.0 3.27 0.0 7 365 365 9,500 8,000 2.42 2.25 2.38 2.0 8 7 7 2,960 2,540 3.10 0.0 4.70 0.0 9 1 3 2,920 2,900 2.00 1.87 2.67 1.50

10 1 33 650 2,660 2.00 1.50 2.38 2.26 11 36 36 960 2,900 2.00 1.62 1.98 1.21 Means k S.D. 59 * 105 71 * 106 3,199 5 2535 3,570 k 1877 2.36 k 0.5 1.42 +- 0.7 3.47 k 1.7 1.18 k 0.8 P N.S. N.S. 0.004 0.001 (Mann-Whitnes U-test)

Thyrocalcitonin Cells in DiGeorge Anomaly 645

control infants. In our study, 80% of DiGeorge cases, and 100% of the control infants had calcitonin immunoreactive cells in their thyroid gland sections. In contrast to Burke et al. [19871, we have only used thyroid gland sections taken from the middle or upper thirds of the lobules. We also measured the volume density of the positive C-cells, since this measurement strongly corre- lates with the calcitonin content of tissue sections [OToole et al., 1985; Wolfe et al., 19751. Furthermore, we analyzed the number of C-cells per thyroid follicule. Considering all of these variables, we confirm the decrease of C-cells in DiGeorge patient’s thyroid glands. Thus, Burke et al.’s work and our study document a reduction in a specific population of cells derived from the cephalic neural crest in patients with DiGeorge anomaly. These findings support the hypothesis of Di- George anomaly as a neurocristopathy.

Finally, counting the C-cells in paraffin-embedded samples could help to elucidate the pathogenesis of malformative conditions, whether clinical or experimental, where cephalic neural crest cell abnormalities are sus- pected (conotruncal cardiac defect [Gamallo et al., 19931, trisomy 18 with hypoplastic thymus, and alco- holic or retinoic acid embryofetopathy).

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dysplasia are urinary system malformations observed in both this and previous series [Conley et al., 19791. However, other reported associated malformations, such as diaphragmatic defects [Conley et al., 19791, thyroid gland hypoplasia [Conley et al., 1979; Robinson, 19751, and abnormal lung lobulation [Moerman et al., 19801 are not present in our patients.

Causal heterogeneity in DiGeorge anomaly was re- viewed by Lammer and Opitz [19861. Although most cases are sporadic and the cause is unknown, several chromosome abnormalities, Mendelian disorders and teratogens may cause this anomaly. Most cases of the DiGeorge anomaly are associated with monosomy for a region of chromosome 22ql1, and DNA markers within the deleted region provide the most sensitive means of detecting this hemizygosity [Scambler et al., 1991, 19921. Families with the DiGeorge anomaly and a pattern of autosomal dominant and autosomal recessive inheritance have been reported; in this sense, the presence of this anomaly in patients with velo-cardio-facial syndrome and Zellweger syndrome is well known. A primary diagnosis of velo-cardio-facial syndrome cannot be ruled out in our patients 2,3 , 7,8, 10, and 11, since many of the anomalies found in this syndrome are not evident a t birth and may not be expressed until late in life. Finally, DiGeorge anomaly has been described in offspring whose mothers were diabetic or exposed to alcohol, retinoids, and/or bisdiamine during pregnancy.

The hypothesis of DiGeorge anomaly as a neurocristopathy was proposed by Bockman and Kirby [1984], who produced DiGeorge-like morphological changes in chick embryos by removing the neural crest. Other experimental studies have shown that the deleterious ef- fects of alcohol and isoretinoin on cephalic neural crest cells may produce the DiGeorge anomaly [Goulding and Pratt, 1986; Sulik et al., 19861.

In the avian and mammalian embryo, C-cells derive from a population of cells which migrate from the rhom- bencephalic neural crest to the ultimobranchial body (fifth brachial pouch) [Le Dourain and Le Litme, 1970; Pearse and Polak, 1971; Polak et al., 19741, which is later incorporated within the thyroid parenchyma. C-cells constitute less than 0.1% of the epithelial cell mass of the thyroid gland [LiVolsi, 19801. These cells have been established as the site of synthesis, storage, and secretion of calcitonin, a peptide hormone that is involved in the homeostatic regulation of the calcium metabolism. Antiserum to calcitonin allows the identi- fication and quantitation of the calcitonin immunoreactive C-cells in the thyroid gland. Thus, C-cells are the only cell subset derived from the cephalic neural crest that may be evaluated in malformative conditions. In human newborn infants, C-cells are preferentially lo- cated in a bilateral zone in the upper two-thirds of the lateral thyroid lobes, where they can be identified indi- vidually and in small groups in both an intrafollicular and parafollicular distribution [Wolfe et al., 19751. C-cells were found to be diminished in the DiGeorge anomaly by Burke et al. [1987], who only considered the presence or absence of C-cells in thyroid sections. Only 27% of the patients studied by these authors had C-cells in their thyroid gland in contrast with 82% of their

646 Palacios et al.

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decrease in thyrocalcitonin-containing cells and analysis of other congenital anomalies in 11...

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