American Journal of Medical Genetics 57:462-471 (1995) Eleven Polish Patients With Microcephaly, Immunodeficiency, and Chromosomal Instability: The Nijmegen Breakage Syndrome K.H. Chrzanowska, W.J. Kleijer, M. Krajewska-Walasek, M. Bialecka, A. Gutkowska, B. Goryluk-Kozakiewicz, J. Michalkiewicz, J. Stachowski, H. Gregorek, G. kyson-Wojciechowska, W. Janowicz, and S. Joiwiak Departments of Genetics (K.H.C., M.K.-W., M.B., A.G., B.G.-K.), Immunology (J.M., H.G.), Clinical Immunology (W.J.), Endocrinology (G.E.- W.), and Neurology (S. J.), Memorial Hospital - Child Health Center, Warsaw, and Second Clinic of Children’s Diseases, University School of Medicine (J.S.), Poznan, Poland; and Department of Clinical Genetics, Erasmus University (W, J.K.), Rotterdam, The Netherlands We report on 11 patients from 8 independent families (3 pairs of sibs) with a complex clin- ical pattern including microcephaly, pecu- liar “bird-like” face, growth retardation, and, in some cases, mild-to-moderate mental deficiency. Most of the patients have recur- ring respiratory tract infections. One girl has developed B-cell lymphoma. A detailed anthropometric study of 15 physical pa- rameters, including 3 cephalic traits, was performed. It was possible to study the chro- mosomes of PHA-stimulated lymphocytes in all of the patients. We found structural aber- rations with multiple rearrangements, pref- erentially involving chromosomes 7 and 14 in a proportion of metaphases in all individ- uals. Profound humoral and cellular im- mune defects were observed. Serum AFP levels were within normal range. Radioresis- tant DNA synthesis was strongly increased in all 8 patients who were hitherto studied in this respect. Our patients fulfill the criteria of the Nijmegen breakage syndrome, which belongs to the growing category of ataxia telangiectasia-related genetic disorders. In light of the increased predisposition to ma- lignancy in this syndrome, an accurate diag- nosis is important for the patient. 0 1995 Wiley-Liss, Inc. KEY WORDS: microcephaly, immunodefi- ciency, chromosome instabil- ity, autosomal-recessive in- Received for publication June 12, 1994; revision received December 9, 1994. Address reprint requests to K.H. Chrzanowska, Department of Genetics, Memorial Hospital-Child Health Center, Al. Dzieci Polskich 20, 04-736 Warsaw, Poland. 0 1995 Wiley-Liss, Inc. heritance, ataxia telangiecta- sia, Nijmegen breakage syn- drome INTRODUCTION The phenomenon of spontaneous chromosome insta- bility was first reported in the mid-1960’s in 3 clearly defined syndromes: Fanconi anemia (FA) [Schroeder et al., 19641,Bloom syndrome (BS) [German et al., 19651, and ataxia telangiectasia (AT) [Hecht et al., 19661. A fourth syndrome, also inherited in an autosomal-reces- sive pattern, was delineated by Weemaes et al. [19811 and was tentatively called the Nijmegen breakage syn- drome (NBS; MIM 251260). Since then, several further patients have been reported [Seemanova et al., 1985; Conley et al., 1986; Wegner et al., 1988; Taalman et al., 1989; Barbi et al., 1991; Bocian et al., 1991; Sanak et al., 1991; Stoppa-Lyonnet et al., 19921. The main clinical manifestations of NBS are: micro- cephaly, peculiar face, stunted growth, and enhanced predisposition to malignancy; intelligence can vary from normal to moderate mental retardation. Such findings as immunodeficiency, chromosome instability with multiple 7 andfor 14 rearrangements, cellular and chromosomal hypersensitivity to X-rays or to the radio- mimetic drug bleomycin, and radioresistance of DNA replication, are common for both NBS and AT. How- ever, NBS patients lack the classical signs and symp- toms of AT, such as cerebellar ataxia, oculocutaneous telangiectasia, and an elevated serum alpha-fetopro- tein (AFP) level. Complementation studies have indicated that pa- tients with NBS are genetically distinct from those with AT. NBS falls into 2 different complementation groups, V1 and V2, whereas AT is represented in 4 sep- arate groups: AB, C, D, and E [Jaspers et al., 1988a,b; Wegner et al., 19881. An important link in understand- ing the relationship between AT and NBS are the twin girls reported by Curry et al. [1989], who had the hall-
Transcript
American Journal of Medical Genetics 57:462-471 (1995)
Eleven Polish Patients With Microcephaly, Immunodeficiency, and Chromosomal Instability: The Nijmegen Breakage Syndrome
K.H. Chrzanowska, W.J. Kleijer, M. Krajewska-Walasek, M. Bialecka, A. Gutkowska, B. Goryluk-Kozakiewicz, J. Michalkiewicz, J. Stachowski, H. Gregorek, G. kyson-Wojciechowska, W. Janowicz, and S. Joiwiak Departments of Genetics (K.H.C., M.K.-W., M.B., A.G., B.G.-K.), Immunology (J.M., H.G.), Clinical Immunology (W.J.), Endocrinology (G.E.- W.), and Neurology (S. J.), Memorial Hospital - Child Health Center, Warsaw, and Second Clinic of Children’s Diseases, University School of Medicine (J.S.), Poznan, Poland; and Department of Clinical Genetics, Erasmus University (W, J.K.), Rotterdam, The Netherlands
We report on 11 patients from 8 independent families (3 pairs of sibs) with a complex clin- ical pattern including microcephaly, pecu- liar “bird-like” face, growth retardation, and, in some cases, mild-to-moderate mental deficiency. Most of the patients have recur- ring respiratory tract infections. One girl has developed B-cell lymphoma. A detailed anthropometric study of 15 physical pa- rameters, including 3 cephalic traits, was performed. It was possible to study the chro- mosomes of PHA-stimulated lymphocytes in all of the patients. We found structural aber- rations with multiple rearrangements, pref- erentially involving chromosomes 7 and 14 in a proportion of metaphases in all individ- uals. Profound humoral and cellular im- mune defects were observed. Serum AFP levels were within normal range. Radioresis- tant DNA synthesis was strongly increased in all 8 patients who were hitherto studied in this respect. Our patients fulfill the criteria of the Nijmegen breakage syndrome, which belongs to the growing category of ataxia telangiectasia-related genetic disorders. In light of the increased predisposition to ma- lignancy in this syndrome, an accurate diag- nosis is important for the patient. 0 1995 Wiley-Liss, Inc.
Received for publication June 12, 1994; revision received December 9, 1994.
Address reprint requests to K.H. Chrzanowska, Department of Genetics, Memorial Hospital-Child Health Center, Al. Dzieci Polskich 20, 04-736 Warsaw, Poland.
INTRODUCTION The phenomenon of spontaneous chromosome insta-
bility was first reported in the mid-1960’s in 3 clearly defined syndromes: Fanconi anemia (FA) [Schroeder et al., 19641, Bloom syndrome (BS) [German et al., 19651, and ataxia telangiectasia (AT) [Hecht e t al., 19661. A fourth syndrome, also inherited in an autosomal-reces- sive pattern, was delineated by Weemaes et al. [19811 and was tentatively called the Nijmegen breakage syn- drome (NBS; MIM 251260). Since then, several further patients have been reported [Seemanova et al., 1985; Conley et al., 1986; Wegner e t al., 1988; Taalman et al., 1989; Barbi et al., 1991; Bocian et al., 1991; Sanak et al., 1991; Stoppa-Lyonnet e t al., 19921.
The main clinical manifestations of NBS are: micro- cephaly, peculiar face, stunted growth, and enhanced predisposition to malignancy; intelligence can vary from normal to moderate mental retardation. Such findings as immunodeficiency, chromosome instability with multiple 7 andfor 14 rearrangements, cellular and chromosomal hypersensitivity to X-rays o r to the radio- mimetic drug bleomycin, and radioresistance of DNA replication, are common for both NBS and AT. How- ever, NBS patients lack the classical signs and symp- toms of AT, such as cerebellar ataxia, oculocutaneous telangiectasia, and an elevated serum alpha-fetopro- tein (AFP) level.
Complementation studies have indicated that pa- tients with NBS are genetically distinct from those with AT. NBS falls into 2 different complementation groups, V1 and V2, whereas AT is represented in 4 sep- arate groups: AB, C, D, and E [Jaspers e t al., 1988a,b; Wegner et al., 19881. An important link in understand- ing the relationship between AT and NBS are the twin girls reported by Curry et al. [1989], who had the hall-
Nijmegen Breakage Syndrome 463
examined under a microscope, after which photographs of all suspected anomalies were systematically taken before the karyotype was established.
Fibroblasts were obtained from skin biopsies. The cul- tures were grown in HAM F10 medium complemented with 10% fetal calf serum, 1% Ultroser G (Villenueve la Garenne, France) (Sepracor S.A., cat no. 2595161, and antibiotics. Chromosomes were prepared from the second or subsequent passages, according to standard methods.
Immunological Studies Total IgG, IgA, and IgM were quantitated by a neph-
elometric method using the Beckman Array Protein System (Beckman Instruments, Inc., Brea, CA).
IgG subclass levels in sera were measured by two- site noncompetitive ELISA. Mouse monoclonal anti- bodies specific for each IgG subclass, and conjugate and standard serum H00-03, were obtained from Janssen Biochimica (Beerse, Belgium). Deficiency of immuno- globulin isotype and IgG subclass was defined as a con- centration more than 2 standard deviations below the age-matched mean.
Cell-mediated immunity was assessed by means of flow cytometric analysis of lymphocyte surface markers (FACScan, Becton-Dickinson, San Jose, CA) and of in vitro response of lymphocytes to PHA, anti-CD3 anti- bodies, anti-TCR antibodies, and Staphylococcus au- reus Cowan I (’H-thymidine incorporation). Production of IL-2, IL-4, and IFN-y by lymphocytes induced with PHA and anti-CD3 antibodies was also studied (ELISA- technique). Results are expressed as mean value i SD.
Biochemical Studies Inhibition of DNA synthesis in cultured skin fibro-
blasts after gamma irradiation was assayed as de- scribed [Jaspers et al., 19901. Cells grown in dishes in Ham’s F10 medium with 15% fetal calf serum, 20 mM HEPES buffer, and antibiotics in a humidified atmos- phere with 3% CO, were prelabeled overnight with [ 14C]-thymidine. After exposure to various doses of gamma rays (137Cs) the cultures were incubated for 4 h r in medium with H-thymidine.
The ratio of 3H and I4C incorporated into DNA was established by dual-label liquid scintillation counting, and the DNA synthesis rate was expressed as a per- centage of the rate in unirradiated cells.
RESULTS Clinical Observations
Characteristics a t birth of all the studied patients are presented in Table I. All the children were born at term; several were small for date. Average birth weight and length were 3,090 g (range 2,300-3,800 g) and 53 cm (range 47-57 cm), respectively. Most of the children were microcephalic from birth.
The clinical manifestations in our 11 patients are summarized in Table 11. At the time of examination all had growth retardation and microcephaly. Those who were born with a normal head circumference had be- come microcephalic a t about 2-3 months of age because of premature closing of the fontanels and sutures. Their facial appearance was very similar and characteristic
3
marks of both syndromes, classical AT and NBS; they were assigned to complementation group V1. The au- thors concluded on these grounds that NBS and these AT variant (AT-V1) patients, referred to as “ATFresno,” do not represent distinct entities but belong t o the spec- trum of one disorder.
Here we evaluate the clinical, cytogenetic, immuno- logical, and biochemical data of our 11 patients who fulfill the diagnostic criteria of NBS.
MATERIALS AND METHODS Patients
Families were ascertained between 1978-1993. Out of over 12,000 patients registered a t the Department of Genetics of the Child Health Center, which is a refer- ence hospital for the entire country, 375 propositi were referred for genetic evaluation of microcephaly and un- derwent cytogenetic screening. Fourteen patients were selected because of structural chromosome aberrations with rearrangements of chromosome pairs 7 and 14; hypogammaglobulinemia had been recognized previ- ously in 2 cases. Three patients seen earlier could not be included in this study because they had developed malignancies: 2 have since died, 1 of leukemia (infor- mation obtained from parents; medical records are not available), 1 of B-cell lymphoma; the third is being treated for IgKappa-positive B-cell lymphoma.
Our study was performed on a final group of 11 pa- tients (8 boys and 3 girls) from 8 families (3 sib pairs and 5 unrelated cases). The abbreviated pedigrees of the families are shown in Figure 1 (families I-VIII). All families are of Polish origin but from different parts of the country; consanguineity either among the families or between the parents was not established. The elder brother of the patient from family VIII died from pneu- monia a t age 9 years, and there are no surviving records, but his mother said he was microcephalic, grew poorly, and was always suffering from severe respiratory infections.
Anthropometric Studies Measurements of 15 somatic, including 3 cephalic,
traits were taken. The data were analyzed using a com- puter program, “BODY,” developed by the Clinical An- thropology Unit of the Child Health Center. The results are presented as diagrams of standard deviations (SDS) according to Polish standards [Kurniewicz-Witczakowa et al., 19831, in respect to both chronological and stat- ural ages (Fig. 4). The statistical significance of differ- ences in somatic and cephalic traits between children with NBS and healthy children of the same chronologi- cal and statural ages was evaluated by Student’s t-test. The degree of significance is marked on the diagrams (from 0.05-0.001).
Cytogenetic Studies Chromosome studies were performed on PHA-stimu-
lated peripheral blood lymphocytes cultured for 72 hr in RPMI-1640 medium supplemented with 20% fetal calf serum and antibiotics. Chromosome analysis was done on metaphases stained with a trypsin-Giemsa banding technique (GTG) according to standard proce- dures. As many cells as possible from each patient were
464 Chrzanowska et al.
ATA f v
F I (K.S.)
‘T r-h m o a f
Fill (K.O. and S.O.)
n I
T hA
ql (Mo.0. and Ma.0.)
rm f
F,,, (ATG. and R.G.)
Fig. 1. Abbreviated pedigrees of 11 patients with NBS (index patients denoted by arrows). F,-,,,,, fam- ily number; 0, male; 0, female; A, abortion.
due to a receding forehead and mandible, prominent midface, upward slant of the palpebral fissures (very much pronounced in some children), and large ears with dysplastic helices (Figs. 2, 3). The typical pheno- type was less distinct in infancy, but a prominent philtrum and receding mandible are evident (Fig. 3d).
Subtle conjunctival telangiectasia was noticed in 5 patients (VK.S., II/Mo.O., II/Ma.O., IV/A.G., and VIII/R.Ch.). A detailed neurological examination of all 11 patients was performed and no signs of ataxia,
apraxic eye movements, or other abnormalities were found. Ophthalmologic examination showed uncharac- teristic “salt and pepper” pigmented fundus in 2 cases.
A predisposition to recurring infections, especially of the respiratory tract, was noted in almost all cases. Pa- tients VIA4.B. and VIII/R.Ch. suffered from chronic bronchopneumonia and have developed bronchiectasis (cystic fibrosis has been excluded). Patient 1K.S. suf- fered from draining ears and repeated episodes of diar- rhea. Slight improvement in both severity and fre-
Nijmegen Breakage Syndrome 465
TABLE I. Birth Data of 11 Patients With NBS
Family
Patient
I K.S.
I1
Mo.0. Ma.0.
111 ~~
K.O. S.O.
Year of birth Sex Number of pregnancy Duration of gestation (weeks) Apgar score Birth weight (g) Birth length (cm) Head circumference (cm) Chest circumference (cm)
1989 M 2
41 10
2,510 50 29 31
1979 1981 F M 1 2
42 42 10 9
3,250 3,250 52 57 30 31.5 34 34
1977 1981 M M 1 3
40 40 9 9
2,300 2,600 50 51 33 31 32 31 -
quency of infections has been noted following intra- venous administration of IgG preparations in these 3 patients. Recurring infections, and even bronchitis andor pneumonia were not so severe in the remaining children.
One boy, VII/K.C., developed autoimmune hemolytic anemia a t age 2 years. The disease was associated with “warm,” class IgG, and “cold,” class IgM, antibodies. He has been receiving treatment for more than 1 year and remains under the care of a hematology unit.
During preparation of this manuscript, a routine semiannual ultrasonographic examination of 1 girl (IIMo.0.) demonstrated presence of an abdominal tu- mor. A specimen taken during laparoscopy was found to be B-cell lymphoma and the patient was being treated accordingly until she died 2 months later. The remain- ing 10 patients are still alive.
Development and Behavior All the children except 1 reached early developmen-
tal milestones in normal time. Delayed speech develop- ment was noticed by parents and confirmed in those children who were tested at an appropriate age. Addi- tionally, some patients needed speech therapy to cor- rect articulation problems.
Psychomotor hyperactivity was evident in infancy and early childhood, while difficulties in concentration became more important later.
I& testing indicated low normal intelligence or bor- derline retardation in half the children (2 and 4, re- spectively). The remaining children were found to be mildly or moderately mentally retarded (4 and 1, re- spectively). Higher verbal than performance I& was es- tablished in most cases. Regardless of I&, the observed children had learning disabilities, especially in subjects requiring abstract reasoning, e.g., mathematics, and their school performance was poorer than could be ex- pected. None of the children have been able to attend normal schools.
All the children have gentle and cheerful personali- ties. Good social interactions are common in spite of shyness. Psychological and emotional problems in- crease with age: depression because of frequent infec- tions, hospitalizations, treatment, andor appearance develops as the children become conscious of their conditions.
The very good physical dexterity of all the children is noteworthy. The boys are especially good in sports
IV
A.G. R.G.
1987 1993 F M 3 4 42 40 10 5
3,050 3,800 47 57 30 33 34 35
V
Mg.0.
1978 F 2 42 7
3,200 52
30.5 35
~
VI
M.B.
1986 M 1
42 10
3,600 56 36 35
VII
K.C.
1991 M 1
39 10
2,950 56 33 32
~
VIII
R.Ch.
1982 M 6
40 10
3,500 52 35 34
activities, including gymnastics, bicycle riding, and soccer, which they like to play very much and in which their performance is no worse than that of their peers.
Anthropometric Studies Analysis of 15 somatic, including 3 cephalic, traits
showed significant growth retardation from the first stages of development, both with respect to chronologi- cal and statural ages (Fig. 4). Height deficiency aver- aged -2.0 SDS, mainly due to shortening of trunk length rather than of lower extremity length, which remained within normal range. Body weight was some- what more deficient and averaged -2.2 SDS. Body weight deficiency was the result of a very small fat com- ponent, while the muscle and bone mass was within normal limits (data not shown). Height and bone age were retarded with respect to calendar age by 2 years and 1YZ years, respectively. Head dimensions (espe- cially circumference, length, and breadth) were even
TABLE 11. Clinical Manifestations in NBS Patients Growth retardation Microcephaly Peculiar face (“bird-like” appearance)
Receding forehead Prominent midface Prominent philtrum Receding mandible Upward slant of palpebral fissures Epicanthic folds Large ears with dysplastic helices
Freckles (mainly in butterfly distribution in face) Areas of hyperpigmentation Areas of hypopigmentation Sun-sensitivity of palpebrae
Skin abnormalities
Telangiectasia (conjunctival) Others
Clinodactyly Polydactyly (preaxial) Transverse palmar crease Wide gap 1/11 toes Syndactyly 111111 toes Renal abnormality Eye fundus with pigment deposits
Out of 652 metaphases analyzed, a total of 202 chro- mosomal rearrangements were detected. The following typical rearrangements involving chromosomes 7 andlor 14 accounted for 65.8% of all abnormalities: inv(7)(p13;q34), 27.2%; t(7;14)(q34;qll), 13.9%; t(7;14) (p13;qll), 8.9%; t(7;7)(p13;q34), 4.0%; and tan(14;14) (qll;q32), 1.0%. Four chromosomal sites, 7p13, 7q34, 14ql1, and 14q32, were preferentially involved in these alterations. Only 2 cases were found in which the breakpoints differed from the sites on chromosome 7 mentioned above. Sometimes more than 1 rearrange- ment per cell was found. Translocations between chro- mosomes 7 or 14 and another chromosome, as well as between 2 chromosomes other than 7 or 14, were ob- served only occasionally.
No rearrangements were found in fibroblasts, al- though chromatid-type instability was observed in the majority of cases. All the primary cultures grew rela- tively slowly, and a t the beginning only epithelioid cells were observed. Actively-migrating fibroblasts did not begin to cover the tissue culture flask until much later (1-3 weeks).
Fig. 2. Patient Mo.0. (a,b) at age 13.5 years, and her brother Ma.0. (c,d) a t age 11.5 years; family 11.
more deficient, ranging from -6.4 to -4.7 SDS in rela- tion to both chronological and statural age. However, proportions among the diminished head measure- ments, i.e., length and breadth, were retained.
Additional analysis of 15 head measurements was performed using a special program (data not shown). Statistically significant differences were found with re- spect to decreased forehead width and shortening of the outer intercanthal distance, whereas the inner inter- canthal distance was within normal range.
Cytogenetic Studies It was possible to study the chromosomes from lym-
phocyte cultures in all 11 patients, although the mitotic index was extremely low and in some cases several re- peats of cultures were needed in order to obtain appro- priate material for chromosomal analysis.
The constitutional karyotypes were all normal, but various structural chromosome aberrations were found in 21.3-46.2% of the metaphases; specific rearrange- ments 9.2-31.3% of cells (Table 111).
chromosomes and l4 were present in Fig. 3. Patient A.G. (a,b) at age 4 years, and her brother R.G. (c,d) at age 4 months; family IV.
DIA
GN
OS
IS :
N
B
S
CH
RO
NO
LOG
ICA
L R
GE
(CA)
SO
MR
TIC
TR
RITS
WE1
GH
T
HE
IGH
T
<L
EN
GT
H>
BO
DY
M
RS
S
IND
EX
TR
UN
K
LE
NG
TH
UP
PE
R
EX
TR
EM
ITY
LE
NG
TH
LOW
ER
E
XT
RE
MIT
Y L
EN
GT
H
BIR
CR
OM
IAL
BR
EA
DT
H
BI I
LIO
CR
IST
RL
E B
RE
RD
TH
CH
ES
T
BR
ER
DT
H
SR
GIT
TR
L D
IAM
ET
ER
O
F
CH
ES
T
CH
ES
T
CIR
CU
MF
ER
EN
CE
AR
M
CIR
CU
MF
ER
EH
CE
TH
IGH
CIR
CU
MF
ER
EN
CE
N=
ll
Me
an
age
(CR
) =
2.6
2
6.
1 cjt-s
D
-2S
D
-1S
D
No
t-n
+
HE
AD
CIR
CU
MF
ER
AN
TE
RO
P
OS
TE
R1
BIP
QR
IET
RL
DIA
HE
TE
R
1 +
2S
D
+3
SD
- 1 ***
j **
* i ***
I*** I ***
I ~ **
* **
* I*** '*
1 ***
I **
i ***
i ***
1 I ***
***
DIF
IGN
OS
IS :
N
B
S
ST
RT
UR
RL
RG
E (SR)
N-1
1
Me
an
age
(SR
) r;
6.6
?
5.1
'J
t-s
SO
MR
TIC
TR
RIT
S
-3S
D
-2S
D
-1S
D
No
t-N
+
1SD
+2
SD
t3
SD
WE
1 GH
T
HE
IGH
T
(LE
NG
TH
)
BO
DY
MlS
SS
IN
DE
X
TR
UN
K
LE
NG
TH
UP
PE
R
EX
TR
EM
ITY
LE
NG
TH
LOW
ER
E
XT
RE
MIT
Y L
EN
GT
H
BIC
ICR
OM
IRL
BR
ER
DT
H
BII
LIO
CR
IST
CIL
E
BR
ER
DT
H
CH
ES
T
BR
ER
DT
H
SC
IGIT
TA
L
DIC
IHE
TE
R
OF
C
HE
Sl
CHEST
CIR
CU
MF
ER
EN
CE
RR
H
CIR
CU
MF
ER
EN
CE
TH
IGH
CIR
CU
MF
ER
EN
CE
HE
RD
C
IRC
UM
FE
RE
NC
E
RN
TE
RO
P
OS
TE
RIO
R
DIR
ME
TE
R
BIP
RR
IET
RL
D
IRM
ET
ER
I I
* -
FJ C
0.0
5,
** -
p
< 0
.01
, X
**
- p
< 0
.00
1
Fig.
4.
Dia
gram
of
SDS
of a
nthr
opom
etri
c m
easu
rem
ents
acc
ordi
ng to
198
3 W
arsa
w S
tand
ards
in
resp
ect t
o ch
rono
logi
cal
age
(CA
) (le
ft) a
nd s
tatu
ral a
ge (S
A) (
righ
t) in
11
chil
dren
wit
h N
BS.
Sta
tura
l ag
e is
the
chro
nolo
gica
l age
at w
hich
the
curr
ent h
eigh
t of a
chi
ld w
ould
cor
resp
ond
to th
e 50
th p
erce
ntil
e.
468 Chrzanowska et al.
TABLE 111. Structural Chromosomal Abnormalities i n Peripheral Blood Lymphocytes of 11 NBS Patients
Other rearrangements than 7 and 14 included; in a few cells there was more than one chromosomal rearrangement.
Immunological Studies shown in Table IVa, which contains only the results of immunoglobulin measurements made before starting
administration). The presence of monoclonal protein
diffusion assays.
The results of quantitative Serum ~mmunog~obu~in
centrations were in 6 children, with an ex-
concentrations were abnormal in all the children. Four I ~ G subclass (I~G,-,) levels were decreased in 1 (1K.S.). A combination of IgG,, IgG,, and IgG, defi- ciency was found in 5 children (II/Ma.O., III/K.o., III/S.O., VI/M.B., and VIII/R.Ch.), IgG, and IgG4 defi- ciency in 3 (II/MO.O., IV/A.G., and v/Mg.o.), and IgG, and IgG4 deficiency in 1 (1Vm.G.). An isolated IgG4 deficiency was found in patient V1UK.C.
The IgA concentration was decreased in 5 children ( IKS . , III/S.O., VI/M.B., VII/K.C., andVIII/R.Ch.). The IgM level was moderately decreased in 2 children (I/K.S. and III/S.O.) and elevated in 1 (V1WK.C.). In this last child, extremely elevated concentrations of IgM were observed in the course of autoimmune hemolytic anemia: 650, 2,130, 1,510, and 991 mg/dl (data not
studies are presented in Table IVa. Low Serum IgG con- any immunomodulating treatment Or immunoglobulin
tremely low value in one of them (1x-S.). IgG subclass was excluded immunoelectroforesis and immune-
Cell-mediated immunity studies are summarized in and CD4-t
cells was generally decreased; only 3 of the 11 patients had a Percentage Of CD3+ T cells. The CD4: CD8 ratio was also decreased in most of the patients. The percentage of B cells was normal in all patients but 1, in whom a dramatic decrease of cells was noted. The proliferative response of T cells t o mitogens (PHA, anti- CD3, and anti-TCR) was decreased in all patients; re- sponse to B cell mitogen (S. aureus Cowan I) was also diminished in all studied patients. Production of cyto- kines (IL-2, IL-4, and IFN-y) by lymphocytes induced with PHA and anti-CD3 was dramatically decreased in all considered patients.
IVb- The percentage Of CD3+
TABLE IVa. Serum Levels of Immunoglobulin Isotgpes, IgG Subclasses, and AFP of 11 NBS Patients"
* It, more than 2 standard deviations below/above age-matched mean. a N 5 8 IUIml.
Nijmegen Breakage Syndrome 469
TABLE IVb. Cell-Mediated Immunity Studies of NBS Patients”’
Patients (N = 11) Controls (N = 4) Phenotypes
% T cells (CD3+) 48 2 17.1 64 2 15 % helper Tc (CD4+) 26 t 13.1 44 t 12 % suppressor Tc (CD8+) 22 i 6 22 5 5 CD4:CD8 ratio 1.2 ? 0.84 2.1 t 0.8 % B cells 5.2 i 3.2 5.8 t- 0.4
PHA 4,179 2 2,599.1 22,932 ? 3,954 Anti-CD3 4,178 i 2,381.1 22,662 t 3,544 Anti-TCR 772 ? 8284 3,954 i 680 S. aureus Cowan I 447 t- 333.1 3,056 ? 1,036
Mitogenic stimulation in cpm ? SD
Cytokines production in pg/ml t- SD 409 ? 1744
267 2 57.1 456 Z 123.1
77 i 26.1 152 t 66.1
1,596 ? 183 86 i 24.1 213 i 1 4
458 2 66 1,509 i 241
202 i 2 466 +- 71
PHA-induced f::: Anti-CD3-induced { E:
IFN-Ly
IFN-Ly
* 1, more than 2 standard deviations below age-matched mean.
Alpha-Fetoprotein (AFP) Studies The serum AFP level in one boy was elevated on first
examination at age 5 months but declined to normal by 15 months; in the remaining patients the AFP levels were within normal range (Table IVa).
Biochemical Studies Radioresistant DNA synthesis (RDS) was assayed in
cultured fibroblasts of 8 patients from 6 families. Fi- broblasts of the remaining 3 patients were growing too poorly for accurate measurements of DNA synthesis. All patients were studied in a series of 3 separate in- vestigations: each investigation included 1 or 2 control cell lines from normal individuals, and 1 or 2 cell lines from previously studied NBS and AT patients, with in- creased levels of RDS.
The clearly-separated shaded areas in Figure 5 rep- resent the DNA synthesis patterns for the 4 normal controls and the 3 NBS/AT patients’ cell lines. The pat- terns for the 8 NBS patients all fall within the NBS/AT area, which demonstrates their increased level of RDS.
DISCUSSION The present 11 children from 8 independent families
share the clinical manifestations of the so-called Nijmegen breakage syndrome [Weemaes et a]., 19811. Significant growth retardation, with respect to both chronological and statural ages, was observed in all the patients from the first stages of development, but the intrauterine growth retardation reported in almost all the patients described earlier [Weemaes et al., 1981; Seemanova et al., 1985; Conley et al., 1986; Wegner et al., 1988; Barbi et al., 19911 was noted in only a few of ours. Microcephaly was also found in all our children, but not from birth in every case, as has been stressed in other studies [Weemaes et al., 1981; Seemanova et al., 1985; Barbi et al., 19911.
In our opinion the phenotype of these patients evolves with age and the typical “bird-like” facial ap- pearance becomes distinct after infancy, as was also observed by Seemanova et al. [1985].
Half the children considered in this study have nor- mal or almost normal (borderline) intelligence and the remainder have been assessed as mentally retarded, mostly to a mild degree. It should be stressed that in all but 1 child, developmental milestones in the first year of life were not delayed. This child has now been found to be moderately retarded. Normal psychomotor devel- opment was reported in some patients [Seemanova et al., 1985; Wegner et al., 1988; Barbi et al., 1991; Bocian et al., 19911; other authors have mentioned mental deficiency of various degrees [Weemaes et al., 1981; Conley et al., 1986; Stoppa-Lyonnet et al., 19921.
Predisposition to recurring infections is caused by a dysfunction of humoral and cellular immunity, con- firmed in all the children, although interpretation of results of 1 boy is difficult due to his young age. Hypo- gammaglobulinemia and defects in IgG subclass distri- bution may depend, among other things, on defects of cytokine synthesis, especially IFN-y, and IL-4, which are responsible for IgGz development and IgGl induc- tion, respectively [Paganelli et al., 19841.
The impaired immune functions in most of the NBS patients, as in AT, involve both T- and B-lymphocytes [Conley et al., 1986; Wegner e t al., 1988; Taalman et al., 1989; Sanak et al., 1991; Stoppa-Lyonnet et al., 1992; present study]. The pattern of immunodeficiency sug- gests impaired maturation of immunocompetent cells. On autopsy, the thymus of several deceased NBS indi- viduals [Seemanova et al., 1985; Weemaes et al., 19911, as in AT [McFarlin et al., 19721, was found to be abnor- mal or even missing. Possible dysfunction of the thy- mus may thus also be a cause of the imbalance between “naive” and “memory” cells, as has been shown in AT patients [Paganelli et al., 19921.
The diminished proliferative response to PHA af- fected the chromosome studies. In some cases several repeats of the PHA-stimulated lymphocyte cultures were required. This phenomenon has also been men- tioned by other authors [Weemaes et al., 1981; Seemanova et al., 1985; Sanak et al., 19911. A high fre- quency of chromosome aberrations involving chromo-
470 Chrzanowska et al.
, + t I I I 0 0 10 15 20
gamma-ray dose (Gy)
J! ig. 3. 1nhibltlOn ot U N A synthesis atter gamma irradiation ot cul- tured skin fibroblasts. -0-, patterns for the 8 NBS patients as indi- cated by their initials; -0-, patterns for 4 normal control individuals, Cl-C4. Shaded areas indicate the intraassay ranges for normal con- trols (as shown for Cl-C4) and for 2 patients with AT and 1 with NBS (individual results not shown).
some bands 7p13,7q34,14qll, and 14q32, the same in both AT and NBS, was observed in all 11 children. The specific breakpoints coincide with the sites of genes having immune functions [Fiorilli et al., 1985; Aurias and Dutrillaux, 19861.
Both immunodeficiency and chromosome instability probably predispose to malignancy (e.g., in FA, 15%, in BS, 4070, and in AT, 20-30% of patients, respectively, develop cancer). Several reported NBS patients also developed neoplasm, mostly of lymphoreticular origin [Seemanova et a]., 1985; Wegner e t al., 1988; Weemaes et al., 19911. Three patients, seen before the present study was undertaken, presented with microcephaly, growth retardation, and chromosome 7 and 14 re- arrangements in a proportion of metaphases, and they developed malignancies at ages 4.5, 5, and 10.5 years, respectively.
In all the present patients, production of cytokines IL-2, IL-4, and IFN-y by lymphocytes induced with PHA and anti-CD3 was significantly decreased. It has been suggested that the lack of IL-2 and IFN-y produc- tion may facilitate the survival of neoplastic cells, since these cytokines are responsible for activation of anti- tumor immunity “losmann and Coffman, 19891. One of the 11 children, a girl, II/Mo.O., developed B-cell lym-
phoma a t age 15 years and died shortly after. I t was not possible to investigate her level of cytokine production a t that time. The remaining patients (the oldest boy is now age 17 years) are carefully monitored for possible development of malignancies.
The 11 patients share a number of important cellular characters with AT, namely a high frequency of chro- mosome 7 and 14 rearrangements with typical break- points, dysfunction of humoral and cellular immunity, and increased resistance of DNA synthesis to gamma- irradiation as demonstrated so far in 8 cases. However, clinically they differ from AT patients. Although in 5 of our patients subtle conjunctival telangiectasia was seen, none of the children examined neurologically had any signs of cerebellar ataxia, apraxic eye movements, or other abnormalities, but all of them presented with microcephaly and a very distinctive phenotype. Also in contrast to AT, the serum AFP levels were normal in all children but one 5-month-old boy. He was retested at age 15 months, and the AFP level was found to be nor- mal. In a girl described by Barbi et al. [1991], serum AFP levels changed from elevated a t age 5 months to normal a t age 2 years. The usual strong decrease of AFP synthesis during pre- and postnatal development may be delayed in NBS; to investigate whether this is a general phenomenon in NBS, we suggest that AFP should be assessed as early as possible.
Recently, a study of simultaneous measurement of both radiosensitivity and defective mitogen response performed by Seyschab et al. [1992], using flow cy- tometry, demonstrated that lymphocytes of NBS patients exhibit high nonproliferating Go-GI fractions, compa- rable to those seen in classical AT. The authors suggest that this method could represent an additional diag- nostic tool.
Another valuable tool to corroborate the diagnosis of NBS or AT is the investigation of the inhibitory effects of irradiation of DNA synthesis in cultured skin fibro- blasts. Nearly all patients reported until now have shown an increased level of RDS, including the 8 NBS patients in the present series. Only a few patients with a clinical diagnosis of AT and normal RDS have been reported [Jaspers et al., 1988a1, whereas increased RDS has been shown in a few patients with atypical forms of AT [Young and Painter, 1989; Willems et al., 19931. This assay of RDS has also been used success- fully for prenatal diagnosis of NBS and AT in the first and second trimester of pregnancy [Jaspers et al., 19901. Reliable prenatal diagnosis, however, requires prior demonstration of clearly increased RDS in fibro- blasts of the affected (index) patient in the family.
We are planning to complete the RDS studies in the fibroblasts of the 3 remaining patients in the present series. Final classification of our patients to one of the complementation groups may contribute to a better un- derstanding of the genetic heterogeneity of this inter- esting syndrome.
An intriguing question still remains open: is the over- representation of European descendants caused by non- random case-finding, as has been suggested by Jaspers et al. [1988al? Our group of patients, as well as others of Polish origin [Bocian et al., 1991; Sanak et al., 19911,
Nijmegen Breakage Syndrome 471
Nijmegen breakage syndrome and ataxia telangiectasia using an assay of radioresistant DNA synthesis. Prenat Diagn 10:667-674.
Kurniewicz-Witczakowa R, Migsowicz I, Niediwiecka Z, Pietrzak M (1983): “Rozw6j fizyczny dzieci i mlodzieiy Warszawskiej.” Warszawa: IMiDz, pp 18-37.
McFarlin DE, Strober W, Waldmann TA (1972): Ataxia telangiectasia. Medicine (Baltimore) 51:281-314.
Mosmann TR, Coffman RL (1989): TH1 and TH2 cells: different pat- terns of lymphokine secretion lead to different functional proper- ties. Annu Rev Immunol 7:145-173.
Paganelli R, Capobianchi MR, Matricardi PM, Cioe L, Seminara R, Dianzani F, Aiuti F (1984): Defective interferon gamma production in ataxia telangiectasia. Clin Immunol Immunopathol32:387-391.
Paganelli R, Scala E, Scarselli E, Ortolani C, Cossarizza A, Carmini D, Aiuti F, Fiorilli M (1992): Selective deficiency of CD4+/ CD45RA+ lymphocytes in patients with ataxia telangiectasia. J Clin Immunol 12:84-91.
Sanak M, Kowalczyk D, Lis G (1991): Congenital immunodeficiency with microcephaly-Seemanova syndrome. Przegl Lek 48:355-357.
Schroeder TM, Anschutz F, Knopp A (1964): Spontane chromosomen- aberrationen bei familiaren panmyelopathie. Humangenetik 1: 194-196.
Seemanova E, Passarge E, BeneSkova D, HouStCk J, Kasal P, Sev- cikova M (1985): Familial microcephaly with normal intelligence, immunodeficiency, and risk for lymphoreticular malignancies: a new autosomal recessive disorder. Am J Med Genet 20:639-648.
Seyschab H, Schindler D, Fried1 R, Barbi G, Boltshauser E, Fryns JP, Hanefeld F, Korinthenberg R, Krageloh-Mann I, Scheres JMJC, Schinzel A, Seemanova E, Tommerup N, Hoehn H (1992): Simul- taneous measurement, using flow cytometry, of radiosensitivity and defective mitogen response in ataxia telangiectasia and re- lated syndromes. Eur J Pediatr 151:756-760.
Stoppa-Lyonnet D, Girault D, LeDeist F, Aurias A (1992): Unusual T cell clones in a patient with Nijmegen breakage syndrome. J Med Genet 29:136-137.
Taalman RDFM, Hustinx TWJ, Weemaes CMR, Seemanova E, Schmidt A, Passarge E, Scheres JMJC (1989): Further delineation ofthe Nijmegen breakage syndrome. Am J Med Genet 32:425-431.
Weemaes CMR, Hustinx TWJ, Scheres JMJC, Van Munster PJJ, Bakkeren JAJM, Taalman RDFM (1981): A new chromosomal instability disorder: the Nijmegen breakage syndrome. Acta Paediatr Scand 70:557-564.
Weemaes CMR, van de Kaa C, Wesseling P, Haraldsson A, Bakkeren J , Seemanova E, Schmidt A, Passarge E (1991): Nijmegen break- age syndrome: clinical, immunological and pathological findings. In Chapel HM, Levinsky RJ , Webster ADB (eds): “Progress in Im- mune Deficiency 111.” London: Royal Society of Medicine Services,
Wegner RD, Metzger M, Hanefeld F, Jaspers NGJ, Baan C, Magdorf K, Kunze J, Sperling K (1988): A new chromosomal instability dis- order confirmed by complementation studies. Clin Genet 3320-32.
Willems PJ, van Roy BC, Kleijer WJ, van der Kraan M, Martin JJ (1993): Atypical clinical presentation of ataxia telangiectasia. Am J Med Genet 45:777-782.
Young BR, Painter RB (1989): Radioresistant DNA synthesis and human genetic diseases. Hum Genet 82:113-117.
Ltd., pp 191-193.
several from the former Czechoslovakia [Seemanova et al., 19851, and single cases described by Conley et al. 119861 and Stoppa-Lyonnet et al. [1992], indicate that Eastern and Central European ancestry is pre- dominant.
ACKNOWLEDGMENTS We thank Mrs. M. van der Kraan and Mr. P. Simons,
Department of Clinical Genetics, Rotterdam, for per- forming DNA synthesis studies in cultured fibroblasts. These studies were supported in part through grant 48405 035 05 from the State Committee for Scientific Research of the Republic of Poland.
REFERENCES Aurias A, Dutrillaux B (1986): Probable involvement of immunoglob-
ulin superfamily genes in most recurrent chromosomal rearrange- ments from ataxia telangiectasia. Hum Genet 72:210-214.
Barbi G, Scheres JMJC, Schindler D, Taalman RDFM, Rodens K, Mehnert K, Muller M, Seyschab H (1991): Chromosome instability and X-ray hypersensitivity in a microcephalic and growth- retarded child. Am J Med Genet 40:44-50.
Bocian E, Obersztyn E, Borowska B (1991): Translocation t(X14)(q28;q11.2) in a patient with clinical symptoms of Nijmegen breakage syndrome. In Vogel W, Jus t W (eds): “3. Tagung der Ges- selschaft fur Humangenetik, Abstracts.” Ulm: Universitatsverlag Ulm, GmbH, p. 169.
Curry CJR, Tsai J, Hutchinson HT, Jaspers NGJ, Wara D, Gatti RA (1989): ATF,,,,,: a phenotype linking ataxia-telangiectasia with the Nijmegan breakage syndrome. Am J Hum Genet 45:270-275.
Fiorilli M, Carbonari M, Crescenzi M, Russo G, Aiuti F (1985): T cell receptor genes and ataxia telangiectasia. Nature 313:186.
German J, Archibald R, Bloom D (1965): Chromosomal breakage in a rare and probably genetically determined syndrome of man. Science 148506-507.
Hecht F, Koler RD, Rigas DA, Dahnke GS, Case MP, Tisdale V, Miller RW (1966): Leukemia and lymphocytes in ataxia telangiectasia. Lancet I1:1193.
Jaspers NGJ, Gatti RA, Baan C, Linssen PCML, Bootsma D (1988a): Genetic complementation analysis of ataxia telangiectasia and Nijmegen breakage syndrome: a survey of 50 patients. Cytogenet Cell Genet 49:259-263.
Jaspers NGJ, Taalman RDFM, Baan C (198813): Patients with an inherited syndrome characterized by immunodeficiency, micro- cephaly, and chromosomal instability: genetic relationship to ataxia telangiectasia. Am J Hum Genet 42:66-73.
Jaspers NGJ, van der Kraan M, Linssen PCML, Maqek M, Seemanova E, Kleijer WJ (1990): First-trimester prenatal diagnosis of the
![[Polish adaptation of swing questionnaire (Survey Work-home Interaction - Nijmegen)]](https://static.documents.pub/doc/80x56/634d4f5b6b2f6dfefb0a49a6/polish-adaptation-of-swing-questionnaire-survey-work-home-interaction-nijmegen.jpg)
