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Late onset 21-hydroxylase deficiency and HLA in the Ashkenazi population: A new Allele at the 21-hydroxylase locus
Late Onset 21-Hydroxylase Deficiency and HLA in the Ashkenazi Population: A New Allele at the 21-Hydroxylase Locus Z. Laron, M.S. Pollack, R. Zamir, A. Roitman, Z. Dickerman, L.S. Levine, F. Lorenzen, G.J. O'Neill, S. Pang, M.I. New, and B. Dupont
A B S T R A C T : The congenital form of 2 l-hydroxylase deficiency (21-OH-def), which results in virilization at birth from intrauterine exposure to testosterone, results from the inheritance of an HLA-linked recessive disease allde from each parent at the 2 l-OH locus (the 21-OH ° alleleS. In this study tce establish a relationship between interntediate attd late onset 21-OH-defar~d HLA. In studies of five Ashkenazi families, we conclude that these syndromes can be caused b~ either of two combinations of genes at the 21-OH locus: They can occur in individua/s who carry the 21-OH ~'allele on one haplotype and who in addition inheril a "susceptibility" 21-OH-def gene (21-OH ~) from their other parent (genotype = 21-OH°/21-OH"): or, they can occur in individuals who are homozygous for the "susceptibility" 2 l-OH-def allele ¢2I-OH*/21 -OHg. The biochemical abnorm~.lity in late onset 21-OH-deficiency is characterized by elevated baseline levels of plasma 17-hydroxyprogesterone (17-OH-P) and urinary pregnanetriol andlor relatiz,ely high 17-OH-P follou,ing A C T H stimulation. Although clinical symptoms are not alu'ays present in all family members with this biochemical abnormality, the ab~mrmality appears to behave as a simple autosomal recessive trait that is linked to HLA. Among the five probands studied, eight of the eight haplotypes from five nonconsang~inous families assumed to carry the 21-OH" allele had the HLA antigens B14 and DRu,I and also had the factor B (Bf) S (Slow) variant. The two remaining disease haplotypes were assu~ued from biochemical data to carry 21-OH" alleles. The results suggest that the biochemical abnormality in these syndromes is linked to HLA and that the 21-OH ~ allele has nonrandom association with the particular HLA haplotype B I 4, DRu, I, B]S in the A~hkenazi population.
ABBREVIATIONS
CAH
congenital adrenal hyperplasia
17-OH-P
21-OH-def
17-hydroxyprogesterone 2 l-hydroxyiase deficiency
Prom the Institute of Pediatric and Adolescent Endocrinolog~ and the Tissue Typing Unit. Beilinson Medical Center, Petah Tiqva, Israel, the Tissue Typing Laboratory. Sloan*Kettering Imtitute for Cancer Research, New York. Neu' York, and the Division of Pediatric Endocrinology, Department of Pediatrics. New York Hospital, Cornell Medical College, New York, New York. Address requestsfor reprints to: Dr. M. S. Pollack, Tissue Typing Laboratory, Sloan-Kette~ingInstitute for CancerResearch, New York. NY 10021. Received •979.
Human lmmunolo~/10 33-66 (t980) © F.l~evierNorth HoUnd, Inc., 1980 52 V~*nderbiltA~ce..New York, NY 10017
55
0198-8859/80101055=! 2 $2.25
56
Z. Laron et ai.
INTRODUCTION Several different forms o f 21-hydroxylase deficiency (21-OH-tieD have now been described. Congenital 2 1 - O H - d e f o r congenital adrenal hyperplasia ( C A H ) due to 2 1 - O H - d e f is known to be an autosomal recessive disease [1] with congenital virilization resulting from increased concentrations o f the precursor steroids 17-hydroxyprogesterone (17-OH-P) and testosterone during intrauterine development. O n e form o f congenital 2 l-OH-clef is also associated with salt wasting due to concomitant aldosterone deficiency. Although the genetic basis for the existence o f these two different forms o f congenital 21-OH-clef has not yet been elucidated, it has been clearly established that both congenital forms are inherited in close linkage to the HLA-B locus o f the human 6th chromosome [ 2 - 7 ~ Detection o f heterozygous carriers o f the 2 1 - O H - d e f allele has been attempted by the use o f A C T H stimulation followed by measurement o f plasma levels o f 17-OH-P [8,9], but the combination o f H L A genotyping and measurement o f ACTH-stimulated plasma 17-OH-P has increased the possibilities for detection o f heterozygous carriers in the families o f patients with 21-OH-def. These carriers almost always have significant elevations in 17-OH-P following A C T H stimuhtion, but are clinically normal [7,10~ A third form o f 2 1 - O H - d e f involving late onset o f symptoms and absence o f salt wasting has also been described [11,12~ This form o f the disease typically involves a female patient with a history o f normal genitalia at birth and normal onset o f puberty followed by the appearance o f mild to severe hirsutism, irregular menses, mild clitoral enlargement in some girls, and increases in urinary and/or plasma androgens. The diagnosis o f 2 l - O H - d e f is established by the presence o f elevated levels o f both basal and ACTH-stimulated 17-OH-P. The two :.atients with this syndrome who were described" by Zachmann and
TABLE 1
Clinical characteristics o f the five probands with late or intermediate onset 21-hydroxylase deficiency
Ethnic Or/gin Father/ Family Mother
Menstrual Data Labial Age Menarche Sex Fusion (yr) (yr)
Hirsudsm
Pretrea~ment Urinary Current Onset Pregnanetrioi Tanner Irregular? Degree (yrs) (mg/24hrY' Stage°
T
Ashkenazi/ Ashkenazi
F
absent 25
15.5
yes
+
14.5
2-8.5
P5
G
Ashkenazi/ Ashkenazi
F
absent 21
12.0
Polymenorrhea
++
16.0
3. i-3.8
P5
Ashkenazi/ Ashkenazi B Spanish/ Ashkenazi
F
absent
18
13.0
Amenorrhea
++
15.0
3-4.4
P5
F
absent
15
i 5.0
Treated
8.5
4-.5.2
P5
L Ashkenazi/ M Ashkenazi
NA"
26
NA
NA
6.5 (pubic)
3.9
P5
O
~blormalvaluesfor urinarypregnaneeriolate alwayslets than2 rag/24horn's. ~Tanne~Stagedesignationsate as degribedby Tarmer(25). "notappficable.
+ HA
Late Onset 2 l-Hydroxylase Deficiency and HLA
57
Prader [12] were considered to be unusually symptomatic heterozygous carriers of the 21-OH" gene since they were related ,o patients with the congenital form of the disease. New et al. [13] have recently reported another case inw~lving the pubertal onset of hirsutism and 21-OH-deficiency. Since the ACTH-stimulated 17-OH-P levels of both parents were normal, and since there were no known relatives affected with congenital 21-OH-def, there was no evidence for the inheritance of a classical 21-OH-def gene in that case. Although it was tentatively concluded on the basis of studying that single family, in which the patient had an unaffected, HLA-identical sister, that the late-onset form of'2 l-OH-def might not be HLA linked [1311, additional studies of this form of the disease were clearly needed. We now report on studies of five additional families; that illustrate the spectrum of clinical and endocrinological abnormalitieg in late. onset 21hydroxylase deficiency. Four of the families have one female patient each with typical late onset 21-hydroxylase deficiency. The fifth family has a male patient ~ith intermediate onset of symptoms of 21-hydroxylase deficiency. The study suggests that these variants of the disease can be genetically explained by assuming that the disease or metabolic abnormality can be caused by different combinations of alleles at the 21-hydroxylase locus, and that the metabolic abnormality is HLA-linked and associated with particulac HLA antigens in the Ashkenazi population. METHODS
Patients and Families. The five patients and their families were selected for this study at the Beil~nsori Medical Center i~ Israel because of intermediate or late presentation of symptoms associated with 21-OH-def but without any other considerations (Table 1). The criteria for diagnosis in girls were absence of labial fusion, generalized hirsutism start'ing close to puberty, marked ache, advanced bone age, menstrual irregularities or amenorrhea and normal or mild clitoral enlargement. The one boy in this series presented with precocious andrenarche, early acne, and advanced bone age. Otherwise, the genitalia in both sexes and gonads in the boy were normal. All patients had elevated urinary pregnanetriol and/or plasma 17-OH-P prior to treatment. These steroids were further increased by ACTH and suppressible by dexamethasone or other glucocorticosteroids. Four of the families were of Ashkenazi extraction and both parents had originated from Central or Eastern Europe (families T,G,O, and L, Table 1). In the fifth family, the mother was Ashkenazi but the father was of Spanish, non-Jewish, Caucasian extraction (family B). Histocompatibility Testing. All available family members and patients in the five families were HLA typed for the well-characterized antigens of the HLA-A,B, and C loci by the standard two-stage microcytotoxicity test utilizing a large battery of established antisera. The families were also typed for the B-cell ailoantigens DRwl-DRw8, using B-lymphocytes isolated after elimination of T-cells passing through a nylon column, and VIII International HLA Workshop reagents were used. The HI.A-linked genetic markers Factor B (Bf) and Glyoxalase I (GLO) were determined by standard procedures [14,15 ]. Endocrine Measurements. The patients' baseline levels of urinary pregnanetriol were measured by standard spectrophotometric techniques [16]. Measurements
58
Z. Laron et al.
TABLE 2
Basal and ACTH-sdmulated levels of 17-hydroxyprogesterone (17-OH-P) in normal and 2 1 - O H ° gene carrier adults Basal Plasma 17-OH-P
Group
Reference
ACTH-Stimulated 17-OH-P (60 rain)
N umber Tested
Range (ng/ml)
Number Tested
Range (ng/ml)
Normal Males
New X'orkHosp.~ Weil et al. [91
14 14
0.2-3.8 1.0-2.3 (1.6~'
4 14
1.8-2.8 1.7-3.8(2.5)~
Normal Females
New York Hosp." Well et al. 191
23 19
0.2-3.8 0.2-3.4 (0.7~'
6 19
1.3-3.2 1-I-4.6 (2.4)~
2 I-OH° Carrier Males
New York Hosp." Well et al. 19]
30 13
0.4- 4.2 0.1-2.8 (1.4)*
8 13
i.4-8.0 3.0-24.7(6.1)I'
21-OH° Carrier Females
New York HospY Well et al. [91
32 24
0.3-4.6 0.3-4.7 (0.7)~
8 24
2.1-8.2 1.7-15.3(4.2)~
~Method$for the meaturement~ffthesevalues are as described in the text. ~MedianvaJuesare indicatedinparentheses.
of baseline and ACTH-stimulated 17-OH-P levels were made in all family members after overnight fasting. The women "arere tested in the early follicular phase (1-7 days after menstruation). Synthetic A C T H 124 (Synacthen®, Ciba), 0.25 mg, was injected over a period of 30 sec as a single bolus. Blood samples were collected at - 1 5 , 0, 30, and 60 mln after injection. The plasma was separated by centrifugation, stored at -200C, and then analyzed for 17-OH-P by radioimmunoassay as previously described [17~ Inter- and intratest variability was less than 12o~. Measured values were analyzed in comparison with 17-OH-P levels measured in a series of normal and heterozygous 21-OH ° gene carrier individuals who were tested with the same technique and also compared with published values (Table 2, [9] and Lorentzen et al. [10] ). Since all the patients and other family members in this study were at least 15 yr of age, 17-OH-P levels measured in adult groups were used for these comparisons. RESULTS The results of HLA genotyl~ing in the five families are shown in Figures 1 and 2 together with a qualitative summary of the 17-OH-P measurements listed in Table 3. The data on Bf allotypes are included in the figures. The data on GLO typing are not shown, but the study did not demonstrate any HLA:GLO recombinations in these families. There were also no intra-HLA recombinations and no HLA:Bf recombinations detected in any o f the families. Each family in the study has a proband (II.1) with the symptoms and signs of intermediate or late onset 21-hydroxylase deficiency summarized in Table 1. N o other patients had been identified in these families prior to this study. In family T (Fig. 1.1), the patient ( I L l ) who has elevated 17-OH-P after A C T H stimulation has an HLA identical brother (11.2) who has slighdy elevated baseline levels of 17-OH-P and very high AC~rH-stimulated 17-OH-P (Table 3) but who is without clinical symptoms. Both l~arents have normal baseline and ACTH-stimulated 17-OH-P levels (Table 3).
i.! Family
I
T
Family
~ 2
a/c
//
alc
~o,~o.... ~
~
c/d
ale
a/d
@
~I-OH-De~
: :;:: :::,:2,: ::~ :22::: 2:: A.3~,814(161
a/b
~o,oo.. ~
~
~l-O~-~ef
c
G
, C-
~
~.33,
i~t4 (.6}
.,.,(.,~, c ~ . ~ , .t~ ( . ~
, DR.I,~fS
o.- .e,s
e,~
(.~
. C -
, DR.I.
.c. c-
o . . , . 8~s , o~.~, a~s o*=,,
af$
e,s
t.3
Family
~
0
-~,i
L~--~ ~0
/ !
c/d
'~:~_~ ~ -
-
_
_
b/d
~. c
/~ ~,~o.... ~
[:~
21-OH-Def ~
~ * ~ . . ~ . ~ ) ~w~.
t
A;~a
*~s
s.~tt.
, c-
, as.t,
Be~
o.-.
a~s
. c-
, De.*,
~ h
(. ~) ..~s(.~,
, el4
o
s
c.~. o ~ . ; , s s
F I G U R E 1 HLA geno:ypes and 17-hydroxyprog,:sterone ¢ i 7-OH-P) levels in families T, G, and O. Baseline a,)d ACTH-stimulated 17-CIH-P levels are qualitatively indicated in the upper and lower squares, respectively (N = in the normal range; single arrow = slight elevation; double arrow = elevation in the 21-OH" gene carrier range; triple arrow = relatively high elevation). Specific wdues for these individuals are listed in Table 3. Control group values are in Table 2. Light: shading in the symbols indicates the presence o f a disease susceptibility allele (21-OH'), according to the genedc model described in the text. Unshaded symbols carry the normal 2 I-OH allele (21oOH"). The b haplotype of the father in family G is inferred to ca;try the same B I 4 and DRwl antigens as the a hapiotype because no other antigens were detected and he is known to be homozygous for HLA Dw I (see text).
2,1
Fomily B
/
O/C
0/¢I
Late Onset 21-OH-Oef
•
Is(.+),
,:-
Ill
, lllllll, , I+4(+i),
~-
a~
l-~.~,
<:-
,l.,~l, &2
¢
~.~
+:-
,
o x . s , I~s Iff
DRill,
, om+l,
I+$
o~.e,
i+~
~oSb
Family
0/c
L
Family
0/¢
O/C
Lo~e Onset
O/d
B
Late Onset
2'~-OH'Oef
Z¢-OH-Oef
~ a.~,~, s~4 ~.s) • ~o , e~. ~.m
, C, c-
, ~.~, , oa.~,
.
. C-
, O+.Z, llS
all+.
lZ
ln<.i)
, I+ 44114) , Cl4,
e~s s~ S
Dm + , + f f
.
~.~.. ~z*,
+ £.IZ,
1+
L
~ c.~) e*~ (.~)
, c, c-
. ~*.~, , ~q.~,
e~s
lSl.
, C-
.011~,
el+
m -.
If+
4I
. 1144114+.
C.4,
e~ ~
FIGURE 2 HLA genotypes and 17-hydroxypro~esterone II.7-OH-P) levels in the families B and L. Symbols for qualitative 17-OH-P levels are as in Fig. I except that N ° indicates values in the overlapping range for normals and 21-OH ° gene carriers. Dark shading indicates the presence of a 21-OH • (null) allele, and light shading indicates the 21-OH s (disease susceptibility) ~tllele. Unshaded symbols represent normal (21-OH a) alleles. In the B family (Fi& 2.1), the pmbaad has inherited one 21-OH ° allele and one 21-OH s allele, according to the genetic model described in the text. Figures 2.2a and 2.2b represent two alternative e>:planatioas for d~e biochemical abnormalities in the L family (see text for details). The i, haplotype of the father in family L is inferred to have the same B14 and D R w l antigens as the ~ haplotype became no other antigens were detected and he is known to be h o m o z y ~ u s for D w l (~ee text).
Late Onset 21-Hydroxylase Deficiency and HLA
61
In family G , (Fig. 1.2), the p a t i e n t : b r o t h e r shares only t h e paternal h a p l o t y p e with the p a t i e n t and has n o r m a l 1 7 - O H - P levels (Table 3): t h e m o t h e r ' s baseline and A C T H - s t i m u l a t e d 1 7 - O H - P levels are also in t h e n o r m a l range (Table 3). A l t h o u g h the father in this family has normal baseline 1 7 - O H - P levels, his 1 7 - O H - P level following A C T H stimulation is h i g h e r than m o s t h e t e r o z g y o u s carriers o f the 2 1 - O H ° allele (Table 3). T h e p a t i e n t in family O has a b r o t h e r w h o differs by b o t h H L A hapaotypes. B o t h the b r o t h e r and the patient's f a t h e r have normal baseline and A C T H s t i m u l a t e d 1 7 - O H - P levels. T h e patient's m o t h e r was unavailable f o r testing. In Figure 1, the i n h e r i t a n c e o f the p r e s u m p t i v e factor that causes late o n s e t 2 1 - O H - d e f in the p r o b a n d s is illustrated by light shading. T h e H L A - i d e n t i c a l b r o t h e r o f the patient in family T is also s h a d e d b e c a u s e he has t h e s a m e b i o c h e m i c a l abnormalities. Since the f a t h e r in family G is the only p a r e n t in t h e s e t h r e e families w h o has h i g h e r levels o f A C T H - s t i m u l a t e d 1 7 - O H - P than m o s t h e t e r o z y g o u s carriers o f 2 1 - O H - d e f , we have also s h a d e d his H L A
T A B L E 3 1 Basal and A C T H - s t i m u l a t e d levels o f 1 7 - h y d r o x y p r o g e s t e r o n e ( 1 7 - O H - P ) in the families o f p a t i e n t s with late o n s e t 2 l - h y d r o x y l a s e deficiency-
Clinical Symptoms?
Basat Plasma 17-OH-P (ng/ml)
Plasma 17-OH-P 60 m:inAfter ACTH-Stimulation (n~mi)
no no yes no
no no yes no
0.9 0.3 2.9 3.2"
2.73 1.98 28.2" 37.8'*
M
no no yes" no
no no yes no
1.5 0.4 17.24" 0.6
14.5b 2.2 88.0* 2.24
M F M
no yes'" no
no yes no
1.2 11.8" 1.2
3.02 I9.0. 1.07
father mother sister sister
M F F F
no no yes" no
no no yes no
1.3 0.5 9.0° 0.5
4.14t' 2.5 23. I a 2.2
father mother brother sister
M F M F
no no yes" no
no no yes no
1.0 0.5 l 8.10 0.5
20.2~ 7.4~' 36.4~ 2.3
Number in Pedigrees (Figs. 1 and 2)
Family Statu,:
Sex
T
1.1 !.2 I1.1 11.2
father mother sister brother
M F F M
G
1. l 1.2 11.1 il.2
father mother sister brother
M F
O
I. 1 I1.1 ll.2
father sister brother
B
i.1 1.2 il. 1 !!.2
L
l. 1 1.2 11.1 11.2
Family
F
Proband?
Methods i'm.the measurement o~ ba.~d and A ~ H - ~ d m u h t ~ 17-OH-P levels ~ e d e . r i n d in t ~ text. ~sults in t ~ ~ ~ c o m ~ with t ~ o b t ~ ~o~ n o r m ~ a ~ 2 1 - O H ° g e ~ c~rier individ~s test~ with t ~ ~ ~ms •~ ~ h ~ s a ~ withp u ~ d v ~ s (~e T ~ 2). Unm~ked v~ues werewitch t ~ norm~ ~n~. ~ v~ e~ ~ fou~ ~ 2 I~H n ~e~ c~r~ers ~ v ~ ~c in t ~ ~ for ~ t e m z y ~ c~Re~ of t ~ 2 l-OH° ~lele (Table2) (seetext for lunar ~ m i ~ ) ~ ~ m s ~ u~er ~ e ~ n t d ~ i ~ me~uremen:sof 17-OH-Plevels
62
Z. Laron et al. haplotype that is not shared with the patient to illustrate the possibility that he may, like the patients, be- homoz~,gous for the genetic disease determinant resulting in late onset 21-OH-def. In family B (Fig. 2.1), the patient's father (I.1) has ACTH-stimulated 17-OH-P levels somewhat higher than normal, and typical of values found among heterozygous carriers of the 21-OH ° allele of classical, congenital 21-OH-def (Table 3). The patient's sister (II.2) who shares the same paternal HLA haplotye, has ACTH-stimulated 17-OH-P levels in the overlapping range of normal females and heteroz~,gous carriers of the 21-OH ° gene (Table 3). Shading in the figure illustrates the possibility that the father's elevated ACTHstimulated 17-OH-P reflects the presence of a 21-OH ° null allele, inherited by both the patient and her sister. The patient has in addition inherited a disease susceptibility factor from the mother. Family L has a patient with intermediate onset of symptoms of 21-OH-def (proband II. 1 of Figs. 2.2a and 2.2b). Since both parents in this family have elevated levels of ACTH-stimulated 17-OH-P, it is possible that the patient simply has a mild form of congenital 21-OH-def which did not express itself for several years and that both parents have elevated 17-OHoP after ACTH stimuhtion because the~' are carriers of the classical 21-OH ° allele (Fig. 2.2a). However, the father's ACTH-stimulated 17-OH-P level is higher than that found among most heterozygous carriers of the classical 2 l-OH-clef gene (Table 3). It is therefore possible that the father in this family, like the patients in the three families shown in Figure 1, is homozygous for the late onset 21-OH-def disease factor. In that case, the patient would have inherited a chssical 2 I-OH" allele from his mother, and a disease susceptibility factor from his father. This possibility is illustrated in Figure 2.2b. The patient's sister has ACTHstimulated 17-OH-P levels consistent with either carrier 2 I-OH" status (Fig. 2.2a) o~, normal status (Fig. 2.2b); in the latter case, her "normal" range 17-OH-P levels could be considered similar to those of the other relatives of patiet~s it; these five families who share a single disease susceptibility haplotype with the patient. The HLA data shown in Figures 1 and 2 also illustrate another major finding in these studies: Eight of the nine HLA haplot~,pes of Ashkenazi Jewish origin that occurred in the probands had the HLA antigens BI4 and DRwl and the Bf variant S. The ninth Ashkenazi haplotype differs from the others in that it is associated with a 21-OH" allele (maternal C haplotype of famillt L, Figs. 2.2a and 2.2b). If the two fathers in families G and L in fact are homozygous for the late onset 21-OH-def susceptibility factor (Figs. 1.2 and 2.2b), we may have identified two additional HLA haplotypes with the BI4,DRwl,BfS haplotype since they can be inferred to be homozygous for B14 and DRwl in the absence of other serologically detected antigens. They are, in fact, known to be homozygous for Dwl from additional studies of in vitro mixed lymphocyte culture reactions in these two families (data not shown). In that case, there are a total of ten such unrelated haplotypes with the 21-OH s allele in these five families. Five of these car~ the A locus allele Aw33 and three carry' A28.
DISCUSSION The spectrum of clinical manifestations and biochemical abnormalities in the late onset 21-hydroxylase deficiency syndromes described in these families can be genetically explained by assuming a relatively simple model. This model utilizes the established fact that the classical congenital 21-h~droxyhse deficiency syndrome is caused by homozygosity for a particular disease allele
Late Onset 21-H~'droxylaseDeficiency,and HLA
6~
(2 I-OH °) at the 21-hydroxylase locus. This locus is closely linked to the HLA-I~ locus [2-7] and is probably located ~etween HLA-B and HLA-D [18]. Heterozygous carriers of the 21-OH ° allele in families of patients with congenital 21-OH-def can be detected by FILA-typing; these individuals almost always show a greater elevation in plasma 17-OH-P levels than normals following ACTH stimulation ([7,10] and Table 2) but are clinically asymptomatic. The HLA data and biochemical data in these five families are consistent with a genetic model that assumes that late onset 21-OH-def can occur in individuals who are homozygous for a different disease allele at the 21-OH locus, a disease susceptibility allele (21-OHS). In the three families illustrated in Figure 1, it is evident that with one exception, the father in family G., heterozygous carriers of this 21-OH s allele do not have significant elevations ir~ ACTH-stimuhted plasma 17-OH-P. A biochemical abnormality correspondit,.g to the genotype 21-OH"/21-OH" cannot be detected by the methods we have used for the determination of baseline levels of plasma 17-OH-P and 17-OFI-P following ACTH stimulation. The HLA-identical brother of one patient (family T), on the other hand, has the identical biochemical abnormalities although he is without clinical symptoms. These data are consistent with a model in which elevation of baseline levels of biochemically abnormal 17-OH-P levels behave as a simple autosomal recessive inherited trait that is linked to HLA. The father in family G could be homozygous, by chance, for the same trait. It appears from this limited study, moreover, that clinical manifestations of the biochemical abnormali~ are more likely to occur in females than in males. Clinical symptoms led to the diagnosis of the disorder in the three female probands, but the HLA identical (male) brother of one patient (family T) and the father of another patient (famBy G) with abno:mal ACTH-stimulated 17-OH-P levels are both without clinical symptoms. The two families illustrated in Figure 2 present another aspect of the genetic model since both families have at least one parent with levels of ACTHstimulated 17-OH-P that fall in the range usually found among heterozygous carriers of the classical 21-O1-[° allele (the father in family B and the mother in family L). In family B, it thus appears that the patient has inherited a 21-OH ° allele from her father and a susceptibility disease determinant (21-OH ~) from her mother who does not have elevated ACTbI-stimulated 17-OH-P. We conclude that late onset 21-OH-def and associated clinical symptoms can occur in individuals with either the genot~pe 21-OFIV21-OH~, as in the families of Figure 1, or with the genotype 21-OH°/21-OH~, as in this family. Family L presents a more complicated situation since both parents actually have elevated ACTH-stimulated 17-OH-P and the patient could have congenital 21-OH-clef with relatively late onset of symptoms, as illustrated in Figure 2.2a. However, the level of the father's ACTH-stimulated 17-OH-P exceeds that found among most heterozygous 21-OH ° gene carriers (see Table 2), and it is possible that his biochemical abnormality, like that of the father in family G (Fig. 1.2) reflects the presence of two 21-OH ~ alleles. This possibility is illustrated in Figure 2.2b, and in this case, the patient would have the genotype 21 -OHS/21 =OH(). The genetic model presented in these figures thus concludes that the biochemical abnormality in late onset 2 l oOH deficiency is expressed in individuals with the genotype 21-OHS/21-OI:'P or in individuals who have one 21-OH s allele and one 21-OH" allele. Linkage of the 21-OH ° allele to HLA has been established by the earlier studies of congenital 21-OH-def [2-7]. Linkage of the 21-OH s allele to HLA cannot yet be established because of the limited number of families studied to date, but the following observations make this the
64
Z. Laron et al. most likel~ conclusion: The only HLA-identi~:al sibling to a patient in any of these five families has the same biochemical abnormality as the patient (family T); no other siblings of patients have significant biochemical abnormalities; the existence of alternative alleles at the same locus is the most likely explanation for effects on the activity of the same enzyme; and, finally, the individuals in this study who carry the 21-OH t allele, all carr~ it in association with the same HLA haplotype: B14,DRwl,BfS. The association of late onset 21d3H-deficiency with a particular HLA haplotype in the Ashkenazi population is another finding of this study. If it is assumed that the fathers in families G and L are homozygous for the B14,DRwl haplotype (see above), 8 out of 8 haplotypes in the probands and 10 out of 10 total haplotypes of Ashkenazi origin that are associated with 21-OH' alleles have the determinants BI4,DRwl and BfS. The average gene frequency for B14 in 9~4 Ashkenazi haplotypes of German, Polish, Russian, and Rumanian extraction can be calculated from the data published by Bonn6-Tamir et al. [19] as 0.118. B14 haplotypes are thus signiftcantly incrvased among the patients ( ~ =48.71) imd among the patients plus biochemically abnormal fathers (X~ = 61.82), and this is statistically signifgant even after correction for the number of antigens being tested. No comparative data is yet available regarding the frequency of DRwl or the B14,DRwl haplotype among control Ashkenazis. It is important to note that the syndrome is associated in the Ashkenazi population wi.-h an HLA haplotype, BI4,DRwI,BfS, rather than with a single HLA determinant. Five of the hapiotypes also have the A locus antigen Aw33. It is possible that this haplotype association is caused by a classical "founder effect." FI6wever, we have also found 21-OH s alleles in association with BI~I haplotypes in two non-Ashkenazi Caucasian families (unpublished observations), ,-nd the geographical and ethnic dispersion of the association suggest that such a "founder effect" would have to be of relatively ancient origin. Because of its association with a particular HLA haplotype, the biochemical abnormality in late onset 21-OH deficiency has a remarkable similarit3, to the autosomal recessive inheritance of deficiency for serum complement C2 which shows strong genetic linkage disequilibrium with the HLA haplotype A25,B18,Dw2,BfS [20,21 ]; Idiopathic bemochromotosis is another disease with incomplete penetrance of clinical symptoms in which a biochemical abnormality is associated with complete HLA haplotypes (A3,B7 or A3,B14) [22,23~ For all three diseases it is thus characteristic that the disease gene is in genetic disequilibrium with one or two HLA haplotypes rather than with one HLA determinant alone. An association was also recently found in congenital 21-OH-def, with the haplotype A3,BwZtT,DRwT,Bt'F (M.S. Pollack, in preparation). Although this disease occurs in association with a large number of other HLA haplotypes [2zt~ the BwZt7 associated disease may represent a distinct variant. This paper demonstrates that the 21-hydroxylase locus can have at least three forms: The normal allele (21-OHn), the classical 21-OH-def allele (21-OH °) and the presently described 21-OH susceptibility allele (21-OHS). The presence of nonrandom gametic association in ,~ariants of both late onse¢ and congenital 21-OH-def suggests that the alleles expressed at the 2 l-hydroxylase locus may relate to the HLA determinants at the functional or molecular level. The 21-OH locus could, therefore, be part of the HLA supergene. Proof of the recessive, monogenic natare of the late onset endocrinological abnormality requires further family studies.
Late Onset 2 l-Hydrox.vlase Deficiency and HLA
65
ACKNOWLEDGMENT This work was supported by the U.S. Publk Health Services, National Institutes of Health, under Grants NCI-CA 22507, CA 17404, CA 08748, CA 19267, EY-01616; by Grant Rl~ 47 from the General Clinical Research Centers Program of the Division of Research Resources, NIH; and by Grant HD 00072.
REFERENCES I. McKusick VA: Mendelian Inheritance in Man, 4th ed. Baltimore, Johns Hopkins Universit.v Press, 1975, p.403. 2. Dupoat B, Oberfield SE, Smithwick EM, Lee TD, Levine LS: Close genetic linkage between HLA and congenital adrenal h.vperplasia (2 l-hydroxylase deficiency). Lancet 2:1309, 1977. 3. Levine IS, Zachmann M, New MI, Prader A, Pollack MS, O'Neill GJ, Yang SY, Oberfield SE, Dupont B: Genetic mapping of ~he 21-hydrox~,lase deficienc,v gene within the HLA linkage group. New Engl J Med 299:911, 1978. 4. Price DA, Klouda PT, Harris R: HLA and congenital adrenal hyperplasia linkage confirmed. Lancet 1:930, 1978. 5. Weitkamp LR, Br.vson M, Bacon GE: HLA and congenital adrenal hyperplasia linkage confirmed. Lancet 1:931, 1978. 6. Puchoit V, Fitzsimmons JS, Gelsthorpe K, Pratt RF, Doughty TW: HLA m~d congenital adrenal h.vperplasia. Lancet 2:1046, 1978. 7. Grosse-Wilde H, Well J, Albert E, Schoiz, S, Bidlingmaier F, Sippel WG, Knorr D: Genetic linkage studies between congenital adrenal h~-perplasia and the HLA blood group s.vstem, lmraunogenetics 8:41, 1979. 8. Gutai Jp, Kowarski AA, Bigeon CJ: The detection of the heteroz,vgous carrier for congenital virilizing adrenal hyperplasia. J Pediatr 90:924, 1977. 9. Well J, Bidlingmaier F, Sippell WG, Butenandt O, Knorr D: Comparison of two tests for heteroz,vgosit~' in congenital adrenal h.vperplasia (CAI-I). Acta Endocrinologica 91:109, 1979. 10. Lorenzen F, Pang S, New MI, Dupont B, Pollack MS, Chow DM, Levine IS: Hormonal phenot~,pe and HLA-,get~ot,vpe in families of patients with congenital adrenal hyperplasia (2 l-hydrox),lase deficienc.v). Pediatr Res 13:1356, 1979. 11. Newmark S, Dluh,v R, Williams G, Pochi P, Rose L; Partial 11 and 21 h.vdroxylase deficiencies in hirsute women. AmJ Obsttyt Gynecol 127:594, 1977. 12. Zachmann M, Prader A: Unusual heteroz.vgotes of congenital adrenal h.vp,~rplasia due to 2 l-h.vdrox.vlase deficienc.v. Acta Endocrinol (Kbh) 87:557, 1978. 13. New M1, Lorenzen F, Pang S, Gunczler P, Dupont B, Levine LS: "'Acquired" adrenal h.vperplasia with 2 [-hydroxylase deficienc.v is not the same genetic disorder as congenital adrenal h.vperplasia. J Clin Endocrinol Metab 48:356, 1979. 14. Alper CA, Boenisch T, Watson L: Genetic polymorphism in human glycine-rich beta-glycoprotein.J Exp Med 135:68, 1972. 15. K~mpfJ, Bissbort S, Ritter H: Red ceil glyoxalase I (E.C:4.4.1.5): Formal genetics and linkage relations. Humangenetik 28:249, 1975. 16. Quazi QH, Hill JG: A modified method for the estimation of pregnanediol, pregnanetriol and seven common 17-ketosteroids in urine by gas liquid chromatography. Steroids 22:311, 1973.
66
Z.I.aron et al. 17. Pang S, Hotchkiss J, Drash AL, Levine I.S, New MI: Microfdter paper method for 17a-hydroxyprogesterone radioimmunoassay: Its application for rapid :;creening for congenital adrenal hyperplasia. J Clin Endocrinoi Metab 45:1003, 1977. 18. P~acholtV, Fitzsimn~onsJS, Reynolds MA, Gelsthorpe K: Location of the gene for 2 l-hydroxylase deficiency (abstr~ts). The European Society for Pediatric Endocrinology. Ulm, September 1979. 19. Bonn6-Tamir B, Bodmer JO, Bodmer WF, Pickbourue P, Brautbar C, Gazit B, Nero S, Zamir R: HLA polymorphism in Israel 9. An overall comparative analysi.q. Tissue Antigens 11:235, 1978. 20. F~aSM, Stern IL Kunkel HG, Dupont B, Hansen JA, Day NK, Good RA, Jersild C, F,3tino M: MLC-determinants and C2 deficiency: LD-Ta associated with C2 deftciency in four families. J Exp Med 142:495, 1975. 21. Raum D, Glass D, Carpenter CB, Aiper CA, Schur PH: The chromosomal order of genes controlling the maior histocompatibility complex, properdi,q factor B, and deficiency of the second component of complement. J Clin Invest 58:12,|0, 1976. 22. Beaumont C, Simon M, Fanchet R, Hespel J-P, Brissot P, Gener.et B. Bourel M: Serum ferritin as a possible marker of the hemochromatosis allele. N Engl J Med 2~ ~.169, 1979. 23. Cartwright GE, Edwards CQ, Kravitz K, Skolnick M, Amos DB, Johnson A, Buskiaer L: Hereditary hemochromatosis. Phenotypic expression of th~ disease. N EnglJ Med 301:175, 1979. 24. Pollack M, Levine L, Zachmann M, Prader A, New M, Oberfield S, Dupont B: Possible genetic linkage disequilibrium between HLA and the 21-hydroxylase deficiency gene (congenital adrenal hyperplasia). Transplant Proc 11:1315, 1979. 25. Tanner JM: Growth at Adolescence, 2nd ed. Blarkweli Scientific Publications, Oxf~,rd, 1962.
A B S T R A C T : The congenital form of 2 l-hydroxylase deficiency (21-OH-def), which results in virilization at birth from intrauterine exposure to testosterone, results from the inheritance of an HLA-linked recessive disease allde from each parent at the 2 l-OH locus (the 21-OH ° alleleS. In this study tce establish a relationship between interntediate attd late onset 21-OH-defar~d HLA. In studies of five Ashkenazi families, we conclude that these syndromes can be caused b~ either of two combinations of genes at the 21-OH locus: They can occur in individua/s who carry the 21-OH ~'allele on one haplotype and who in addition inheril a "susceptibility" 21-OH-def gene (21-OH ~) from their other parent (genotype = 21-OH°/21-OH"): or, they can occur in individuals who are homozygous for the "susceptibility" 2 l-OH-def allele ¢2I-OH*/21 -OHg. The biochemical abnorm~.lity in late onset 21-OH-deficiency is characterized by elevated baseline levels of plasma 17-hydroxyprogesterone (17-OH-P) and urinary pregnanetriol andlor relatiz,ely high 17-OH-P follou,ing A C T H stimulation. Although clinical symptoms are not alu'ays present in all family members with this biochemical abnormality, the ab~mrmality appears to behave as a simple autosomal recessive trait that is linked to HLA. Among the five probands studied, eight of the eight haplotypes from five nonconsang~inous families assumed to carry the 21-OH" allele had the HLA antigens B14 and DRu,I and also had the factor B (Bf) S (Slow) variant. The two remaining disease haplotypes were assu~ued from biochemical data to carry 21-OH" alleles. The results suggest that the biochemical abnormality in these syndromes is linked to HLA and that the 21-OH ~ allele has nonrandom association with the particular HLA haplotype B I 4, DRu, I, B]S in the A~hkenazi population.
ABBREVIATIONS
CAH
congenital adrenal hyperplasia
17-OH-P
21-OH-def
17-hydroxyprogesterone 2 l-hydroxyiase deficiency
Prom the Institute of Pediatric and Adolescent Endocrinolog~ and the Tissue Typing Unit. Beilinson Medical Center, Petah Tiqva, Israel, the Tissue Typing Laboratory. Sloan*Kettering Imtitute for Cancer Research, New York. Neu' York, and the Division of Pediatric Endocrinology, Department of Pediatrics. New York Hospital, Cornell Medical College, New York, New York. Address requestsfor reprints to: Dr. M. S. Pollack, Tissue Typing Laboratory, Sloan-Kette~ingInstitute for CancerResearch, New York. NY 10021. Received •979.
Human lmmunolo~/10 33-66 (t980) © F.l~evierNorth HoUnd, Inc., 1980 52 V~*nderbiltA~ce..New York, NY 10017
55
0198-8859/80101055=! 2 $2.25
56
Z. Laron et ai.
INTRODUCTION Several different forms o f 21-hydroxylase deficiency (21-OH-tieD have now been described. Congenital 2 1 - O H - d e f o r congenital adrenal hyperplasia ( C A H ) due to 2 1 - O H - d e f is known to be an autosomal recessive disease [1] with congenital virilization resulting from increased concentrations o f the precursor steroids 17-hydroxyprogesterone (17-OH-P) and testosterone during intrauterine development. O n e form o f congenital 2 l-OH-clef is also associated with salt wasting due to concomitant aldosterone deficiency. Although the genetic basis for the existence o f these two different forms o f congenital 21-OH-clef has not yet been elucidated, it has been clearly established that both congenital forms are inherited in close linkage to the HLA-B locus o f the human 6th chromosome [ 2 - 7 ~ Detection o f heterozygous carriers o f the 2 1 - O H - d e f allele has been attempted by the use o f A C T H stimulation followed by measurement o f plasma levels o f 17-OH-P [8,9], but the combination o f H L A genotyping and measurement o f ACTH-stimulated plasma 17-OH-P has increased the possibilities for detection o f heterozygous carriers in the families o f patients with 21-OH-def. These carriers almost always have significant elevations in 17-OH-P following A C T H stimuhtion, but are clinically normal [7,10~ A third form o f 2 1 - O H - d e f involving late onset o f symptoms and absence o f salt wasting has also been described [11,12~ This form o f the disease typically involves a female patient with a history o f normal genitalia at birth and normal onset o f puberty followed by the appearance o f mild to severe hirsutism, irregular menses, mild clitoral enlargement in some girls, and increases in urinary and/or plasma androgens. The diagnosis o f 2 l - O H - d e f is established by the presence o f elevated levels o f both basal and ACTH-stimulated 17-OH-P. The two :.atients with this syndrome who were described" by Zachmann and
TABLE 1
Clinical characteristics o f the five probands with late or intermediate onset 21-hydroxylase deficiency
Ethnic Or/gin Father/ Family Mother
Menstrual Data Labial Age Menarche Sex Fusion (yr) (yr)
Hirsudsm
Pretrea~ment Urinary Current Onset Pregnanetrioi Tanner Irregular? Degree (yrs) (mg/24hrY' Stage°
T
Ashkenazi/ Ashkenazi
F
absent 25
15.5
yes
+
14.5
2-8.5
P5
G
Ashkenazi/ Ashkenazi
F
absent 21
12.0
Polymenorrhea
++
16.0
3. i-3.8
P5
Ashkenazi/ Ashkenazi B Spanish/ Ashkenazi
F
absent
18
13.0
Amenorrhea
++
15.0
3-4.4
P5
F
absent
15
i 5.0
Treated
8.5
4-.5.2
P5
L Ashkenazi/ M Ashkenazi
NA"
26
NA
NA
6.5 (pubic)
3.9
P5
O
~blormalvaluesfor urinarypregnaneeriolate alwayslets than2 rag/24horn's. ~Tanne~Stagedesignationsate as degribedby Tarmer(25). "notappficable.
+ HA
Late Onset 2 l-Hydroxylase Deficiency and HLA
57
Prader [12] were considered to be unusually symptomatic heterozygous carriers of the 21-OH" gene since they were related ,o patients with the congenital form of the disease. New et al. [13] have recently reported another case inw~lving the pubertal onset of hirsutism and 21-OH-deficiency. Since the ACTH-stimulated 17-OH-P levels of both parents were normal, and since there were no known relatives affected with congenital 21-OH-def, there was no evidence for the inheritance of a classical 21-OH-def gene in that case. Although it was tentatively concluded on the basis of studying that single family, in which the patient had an unaffected, HLA-identical sister, that the late-onset form of'2 l-OH-def might not be HLA linked [1311, additional studies of this form of the disease were clearly needed. We now report on studies of five additional families; that illustrate the spectrum of clinical and endocrinological abnormalitieg in late. onset 21hydroxylase deficiency. Four of the families have one female patient each with typical late onset 21-hydroxylase deficiency. The fifth family has a male patient ~ith intermediate onset of symptoms of 21-hydroxylase deficiency. The study suggests that these variants of the disease can be genetically explained by assuming that the disease or metabolic abnormality can be caused by different combinations of alleles at the 21-hydroxylase locus, and that the metabolic abnormality is HLA-linked and associated with particulac HLA antigens in the Ashkenazi population. METHODS
Patients and Families. The five patients and their families were selected for this study at the Beil~nsori Medical Center i~ Israel because of intermediate or late presentation of symptoms associated with 21-OH-def but without any other considerations (Table 1). The criteria for diagnosis in girls were absence of labial fusion, generalized hirsutism start'ing close to puberty, marked ache, advanced bone age, menstrual irregularities or amenorrhea and normal or mild clitoral enlargement. The one boy in this series presented with precocious andrenarche, early acne, and advanced bone age. Otherwise, the genitalia in both sexes and gonads in the boy were normal. All patients had elevated urinary pregnanetriol and/or plasma 17-OH-P prior to treatment. These steroids were further increased by ACTH and suppressible by dexamethasone or other glucocorticosteroids. Four of the families were of Ashkenazi extraction and both parents had originated from Central or Eastern Europe (families T,G,O, and L, Table 1). In the fifth family, the mother was Ashkenazi but the father was of Spanish, non-Jewish, Caucasian extraction (family B). Histocompatibility Testing. All available family members and patients in the five families were HLA typed for the well-characterized antigens of the HLA-A,B, and C loci by the standard two-stage microcytotoxicity test utilizing a large battery of established antisera. The families were also typed for the B-cell ailoantigens DRwl-DRw8, using B-lymphocytes isolated after elimination of T-cells passing through a nylon column, and VIII International HLA Workshop reagents were used. The HI.A-linked genetic markers Factor B (Bf) and Glyoxalase I (GLO) were determined by standard procedures [14,15 ]. Endocrine Measurements. The patients' baseline levels of urinary pregnanetriol were measured by standard spectrophotometric techniques [16]. Measurements
58
Z. Laron et al.
TABLE 2
Basal and ACTH-sdmulated levels of 17-hydroxyprogesterone (17-OH-P) in normal and 2 1 - O H ° gene carrier adults Basal Plasma 17-OH-P
Group
Reference
ACTH-Stimulated 17-OH-P (60 rain)
N umber Tested
Range (ng/ml)
Number Tested
Range (ng/ml)
Normal Males
New X'orkHosp.~ Weil et al. [91
14 14
0.2-3.8 1.0-2.3 (1.6~'
4 14
1.8-2.8 1.7-3.8(2.5)~
Normal Females
New York Hosp." Well et al. 191
23 19
0.2-3.8 0.2-3.4 (0.7~'
6 19
1.3-3.2 1-I-4.6 (2.4)~
2 I-OH° Carrier Males
New York Hosp." Well et al. 19]
30 13
0.4- 4.2 0.1-2.8 (1.4)*
8 13
i.4-8.0 3.0-24.7(6.1)I'
21-OH° Carrier Females
New York HospY Well et al. [91
32 24
0.3-4.6 0.3-4.7 (0.7)~
8 24
2.1-8.2 1.7-15.3(4.2)~
~Method$for the meaturement~ffthesevalues are as described in the text. ~MedianvaJuesare indicatedinparentheses.
of baseline and ACTH-stimulated 17-OH-P levels were made in all family members after overnight fasting. The women "arere tested in the early follicular phase (1-7 days after menstruation). Synthetic A C T H 124 (Synacthen®, Ciba), 0.25 mg, was injected over a period of 30 sec as a single bolus. Blood samples were collected at - 1 5 , 0, 30, and 60 mln after injection. The plasma was separated by centrifugation, stored at -200C, and then analyzed for 17-OH-P by radioimmunoassay as previously described [17~ Inter- and intratest variability was less than 12o~. Measured values were analyzed in comparison with 17-OH-P levels measured in a series of normal and heterozygous 21-OH ° gene carrier individuals who were tested with the same technique and also compared with published values (Table 2, [9] and Lorentzen et al. [10] ). Since all the patients and other family members in this study were at least 15 yr of age, 17-OH-P levels measured in adult groups were used for these comparisons. RESULTS The results of HLA genotyl~ing in the five families are shown in Figures 1 and 2 together with a qualitative summary of the 17-OH-P measurements listed in Table 3. The data on Bf allotypes are included in the figures. The data on GLO typing are not shown, but the study did not demonstrate any HLA:GLO recombinations in these families. There were also no intra-HLA recombinations and no HLA:Bf recombinations detected in any o f the families. Each family in the study has a proband (II.1) with the symptoms and signs of intermediate or late onset 21-hydroxylase deficiency summarized in Table 1. N o other patients had been identified in these families prior to this study. In family T (Fig. 1.1), the patient ( I L l ) who has elevated 17-OH-P after A C T H stimulation has an HLA identical brother (11.2) who has slighdy elevated baseline levels of 17-OH-P and very high AC~rH-stimulated 17-OH-P (Table 3) but who is without clinical symptoms. Both l~arents have normal baseline and ACTH-stimulated 17-OH-P levels (Table 3).
i.! Family
I
T
Family
~ 2
a/c
//
alc
~o,~o.... ~
~
c/d
ale
a/d
@
~I-OH-De~
: :;:: :::,:2,: ::~ :22::: 2:: A.3~,814(161
a/b
~o,oo.. ~
~
~l-O~-~ef
c
G
, C-
~
~.33,
i~t4 (.6}
.,.,(.,~, c ~ . ~ , .t~ ( . ~
, DR.I,~fS
o.- .e,s
e,~
(.~
. C -
, DR.I.
.c. c-
o . . , . 8~s , o~.~, a~s o*=,,
af$
e,s
t.3
Family
~
0
-~,i
L~--~ ~0
/ !
c/d
'~:~_~ ~ -
-
_
_
b/d
~. c
/~ ~,~o.... ~
[:~
21-OH-Def ~
~ * ~ . . ~ . ~ ) ~w~.
t
A;~a
*~s
s.~tt.
, c-
, as.t,
Be~
o.-.
a~s
. c-
, De.*,
~ h
(. ~) ..~s(.~,
, el4
o
s
c.~. o ~ . ; , s s
F I G U R E 1 HLA geno:ypes and 17-hydroxyprog,:sterone ¢ i 7-OH-P) levels in families T, G, and O. Baseline a,)d ACTH-stimulated 17-CIH-P levels are qualitatively indicated in the upper and lower squares, respectively (N = in the normal range; single arrow = slight elevation; double arrow = elevation in the 21-OH" gene carrier range; triple arrow = relatively high elevation). Specific wdues for these individuals are listed in Table 3. Control group values are in Table 2. Light: shading in the symbols indicates the presence o f a disease susceptibility allele (21-OH'), according to the genedc model described in the text. Unshaded symbols carry the normal 2 I-OH allele (21oOH"). The b haplotype of the father in family G is inferred to ca;try the same B I 4 and DRwl antigens as the a hapiotype because no other antigens were detected and he is known to be homozygous for HLA Dw I (see text).
2,1
Fomily B
/
O/C
0/¢I
Late Onset 21-OH-Oef
•
Is(.+),
,:-
Ill
, lllllll, , I+4(+i),
~-
a~
l-~.~,
<:-
,l.,~l, &2
¢
~.~
+:-
,
o x . s , I~s Iff
DRill,
, om+l,
I+$
o~.e,
i+~
~oSb
Family
0/c
L
Family
0/¢
O/C
Lo~e Onset
O/d
B
Late Onset
2'~-OH'Oef
Z¢-OH-Oef
~ a.~,~, s~4 ~.s) • ~o , e~. ~.m
, C, c-
, ~.~, , oa.~,
.
. C-
, O+.Z, llS
all+.
lZ
ln<.i)
, I+ 44114) , Cl4,
e~s s~ S
Dm + , + f f
.
~.~.. ~z*,
+ £.IZ,
1+
L
~ c.~) e*~ (.~)
, c, c-
. ~*.~, , ~q.~,
e~s
lSl.
, C-
.011~,
el+
m -.
If+
4I
. 1144114+.
C.4,
e~ ~
FIGURE 2 HLA genotypes and 17-hydroxypro~esterone II.7-OH-P) levels in the families B and L. Symbols for qualitative 17-OH-P levels are as in Fig. I except that N ° indicates values in the overlapping range for normals and 21-OH ° gene carriers. Dark shading indicates the presence of a 21-OH • (null) allele, and light shading indicates the 21-OH s (disease susceptibility) ~tllele. Unshaded symbols represent normal (21-OH a) alleles. In the B family (Fi& 2.1), the pmbaad has inherited one 21-OH ° allele and one 21-OH s allele, according to the genetic model described in the text. Figures 2.2a and 2.2b represent two alternative e>:planatioas for d~e biochemical abnormalities in the L family (see text for details). The i, haplotype of the father in family L is inferred to have the same B14 and D R w l antigens as the ~ haplotype became no other antigens were detected and he is known to be h o m o z y ~ u s for D w l (~ee text).
Late Onset 21-Hydroxylase Deficiency and HLA
61
In family G , (Fig. 1.2), the p a t i e n t : b r o t h e r shares only t h e paternal h a p l o t y p e with the p a t i e n t and has n o r m a l 1 7 - O H - P levels (Table 3): t h e m o t h e r ' s baseline and A C T H - s t i m u l a t e d 1 7 - O H - P levels are also in t h e n o r m a l range (Table 3). A l t h o u g h the father in this family has normal baseline 1 7 - O H - P levels, his 1 7 - O H - P level following A C T H stimulation is h i g h e r than m o s t h e t e r o z g y o u s carriers o f the 2 1 - O H ° allele (Table 3). T h e p a t i e n t in family O has a b r o t h e r w h o differs by b o t h H L A hapaotypes. B o t h the b r o t h e r and the patient's f a t h e r have normal baseline and A C T H s t i m u l a t e d 1 7 - O H - P levels. T h e patient's m o t h e r was unavailable f o r testing. In Figure 1, the i n h e r i t a n c e o f the p r e s u m p t i v e factor that causes late o n s e t 2 1 - O H - d e f in the p r o b a n d s is illustrated by light shading. T h e H L A - i d e n t i c a l b r o t h e r o f the patient in family T is also s h a d e d b e c a u s e he has t h e s a m e b i o c h e m i c a l abnormalities. Since the f a t h e r in family G is the only p a r e n t in t h e s e t h r e e families w h o has h i g h e r levels o f A C T H - s t i m u l a t e d 1 7 - O H - P than m o s t h e t e r o z y g o u s carriers o f 2 1 - O H - d e f , we have also s h a d e d his H L A
T A B L E 3 1 Basal and A C T H - s t i m u l a t e d levels o f 1 7 - h y d r o x y p r o g e s t e r o n e ( 1 7 - O H - P ) in the families o f p a t i e n t s with late o n s e t 2 l - h y d r o x y l a s e deficiency-
Clinical Symptoms?
Basat Plasma 17-OH-P (ng/ml)
Plasma 17-OH-P 60 m:inAfter ACTH-Stimulation (n~mi)
no no yes no
no no yes no
0.9 0.3 2.9 3.2"
2.73 1.98 28.2" 37.8'*
M
no no yes" no
no no yes no
1.5 0.4 17.24" 0.6
14.5b 2.2 88.0* 2.24
M F M
no yes'" no
no yes no
1.2 11.8" 1.2
3.02 I9.0. 1.07
father mother sister sister
M F F F
no no yes" no
no no yes no
1.3 0.5 9.0° 0.5
4.14t' 2.5 23. I a 2.2
father mother brother sister
M F M F
no no yes" no
no no yes no
1.0 0.5 l 8.10 0.5
20.2~ 7.4~' 36.4~ 2.3
Number in Pedigrees (Figs. 1 and 2)
Family Statu,:
Sex
T
1.1 !.2 I1.1 11.2
father mother sister brother
M F F M
G
1. l 1.2 11.1 il.2
father mother sister brother
M F
O
I. 1 I1.1 ll.2
father sister brother
B
i.1 1.2 il. 1 !!.2
L
l. 1 1.2 11.1 11.2
Family
F
Proband?
Methods i'm.the measurement o~ ba.~d and A ~ H - ~ d m u h t ~ 17-OH-P levels ~ e d e . r i n d in t ~ text. ~sults in t ~ ~ ~ c o m ~ with t ~ o b t ~ ~o~ n o r m ~ a ~ 2 1 - O H ° g e ~ c~rier individ~s test~ with t ~ ~ ~ms •~ ~ h ~ s a ~ withp u ~ d v ~ s (~e T ~ 2). Unm~ked v~ues werewitch t ~ norm~ ~n~. ~ v~ e~ ~ fou~ ~ 2 I~H n ~e~ c~r~ers ~ v ~ ~c in t ~ ~ for ~ t e m z y ~ c~Re~ of t ~ 2 l-OH° ~lele (Table2) (seetext for lunar ~ m i ~ ) ~ ~ m s ~ u~er ~ e ~ n t d ~ i ~ me~uremen:sof 17-OH-Plevels
62
Z. Laron et al. haplotype that is not shared with the patient to illustrate the possibility that he may, like the patients, be- homoz~,gous for the genetic disease determinant resulting in late onset 21-OH-def. In family B (Fig. 2.1), the patient's father (I.1) has ACTH-stimulated 17-OH-P levels somewhat higher than normal, and typical of values found among heterozygous carriers of the 21-OH ° allele of classical, congenital 21-OH-def (Table 3). The patient's sister (II.2) who shares the same paternal HLA haplotye, has ACTH-stimulated 17-OH-P levels in the overlapping range of normal females and heteroz~,gous carriers of the 21-OH ° gene (Table 3). Shading in the figure illustrates the possibility that the father's elevated ACTHstimulated 17-OH-P reflects the presence of a 21-OH ° null allele, inherited by both the patient and her sister. The patient has in addition inherited a disease susceptibility factor from the mother. Family L has a patient with intermediate onset of symptoms of 21-OH-def (proband II. 1 of Figs. 2.2a and 2.2b). Since both parents in this family have elevated levels of ACTH-stimulated 17-OH-P, it is possible that the patient simply has a mild form of congenital 21-OH-def which did not express itself for several years and that both parents have elevated 17-OHoP after ACTH stimuhtion because the~' are carriers of the classical 21-OH ° allele (Fig. 2.2a). However, the father's ACTH-stimulated 17-OH-P level is higher than that found among most heterozygous carriers of the classical 2 l-OH-clef gene (Table 3). It is therefore possible that the father in this family, like the patients in the three families shown in Figure 1, is homozygous for the late onset 21-OH-def disease factor. In that case, the patient would have inherited a chssical 2 I-OH" allele from his mother, and a disease susceptibility factor from his father. This possibility is illustrated in Figure 2.2b. The patient's sister has ACTHstimulated 17-OH-P levels consistent with either carrier 2 I-OH" status (Fig. 2.2a) o~, normal status (Fig. 2.2b); in the latter case, her "normal" range 17-OH-P levels could be considered similar to those of the other relatives of patiet~s it; these five families who share a single disease susceptibility haplotype with the patient. The HLA data shown in Figures 1 and 2 also illustrate another major finding in these studies: Eight of the nine HLA haplot~,pes of Ashkenazi Jewish origin that occurred in the probands had the HLA antigens BI4 and DRwl and the Bf variant S. The ninth Ashkenazi haplotype differs from the others in that it is associated with a 21-OH" allele (maternal C haplotype of famillt L, Figs. 2.2a and 2.2b). If the two fathers in families G and L in fact are homozygous for the late onset 21-OH-def susceptibility factor (Figs. 1.2 and 2.2b), we may have identified two additional HLA haplotypes with the BI4,DRwl,BfS haplotype since they can be inferred to be homozygous for B14 and DRwl in the absence of other serologically detected antigens. They are, in fact, known to be homozygous for Dwl from additional studies of in vitro mixed lymphocyte culture reactions in these two families (data not shown). In that case, there are a total of ten such unrelated haplotypes with the 21-OH s allele in these five families. Five of these car~ the A locus allele Aw33 and three carry' A28.
DISCUSSION The spectrum of clinical manifestations and biochemical abnormalities in the late onset 21-hydroxylase deficiency syndromes described in these families can be genetically explained by assuming a relatively simple model. This model utilizes the established fact that the classical congenital 21-h~droxyhse deficiency syndrome is caused by homozygosity for a particular disease allele
Late Onset 21-H~'droxylaseDeficiency,and HLA
6~
(2 I-OH °) at the 21-hydroxylase locus. This locus is closely linked to the HLA-I~ locus [2-7] and is probably located ~etween HLA-B and HLA-D [18]. Heterozygous carriers of the 21-OH ° allele in families of patients with congenital 21-OH-def can be detected by FILA-typing; these individuals almost always show a greater elevation in plasma 17-OH-P levels than normals following ACTH stimulation ([7,10] and Table 2) but are clinically asymptomatic. The HLA data and biochemical data in these five families are consistent with a genetic model that assumes that late onset 21-OH-def can occur in individuals who are homozygous for a different disease allele at the 21-OH locus, a disease susceptibility allele (21-OHS). In the three families illustrated in Figure 1, it is evident that with one exception, the father in family G., heterozygous carriers of this 21-OH s allele do not have significant elevations ir~ ACTH-stimuhted plasma 17-OH-P. A biochemical abnormality correspondit,.g to the genotype 21-OH"/21-OH" cannot be detected by the methods we have used for the determination of baseline levels of plasma 17-OH-P and 17-OFI-P following ACTH stimulation. The HLA-identical brother of one patient (family T), on the other hand, has the identical biochemical abnormalities although he is without clinical symptoms. These data are consistent with a model in which elevation of baseline levels of biochemically abnormal 17-OH-P levels behave as a simple autosomal recessive inherited trait that is linked to HLA. The father in family G could be homozygous, by chance, for the same trait. It appears from this limited study, moreover, that clinical manifestations of the biochemical abnormali~ are more likely to occur in females than in males. Clinical symptoms led to the diagnosis of the disorder in the three female probands, but the HLA identical (male) brother of one patient (family T) and the father of another patient (famBy G) with abno:mal ACTH-stimulated 17-OH-P levels are both without clinical symptoms. The two families illustrated in Figure 2 present another aspect of the genetic model since both families have at least one parent with levels of ACTHstimulated 17-OH-P that fall in the range usually found among heterozygous carriers of the classical 21-O1-[° allele (the father in family B and the mother in family L). In family B, it thus appears that the patient has inherited a 21-OH ° allele from her father and a susceptibility disease determinant (21-OH ~) from her mother who does not have elevated ACTbI-stimulated 17-OH-P. We conclude that late onset 21-OH-def and associated clinical symptoms can occur in individuals with either the genot~pe 21-OFIV21-OH~, as in the families of Figure 1, or with the genotype 21-OH°/21-OH~, as in this family. Family L presents a more complicated situation since both parents actually have elevated ACTH-stimulated 17-OH-P and the patient could have congenital 21-OH-clef with relatively late onset of symptoms, as illustrated in Figure 2.2a. However, the level of the father's ACTH-stimulated 17-OH-P exceeds that found among most heterozygous 21-OH ° gene carriers (see Table 2), and it is possible that his biochemical abnormality, like that of the father in family G (Fig. 1.2) reflects the presence of two 21-OH ~ alleles. This possibility is illustrated in Figure 2.2b, and in this case, the patient would have the genotype 21 -OHS/21 =OH(). The genetic model presented in these figures thus concludes that the biochemical abnormality in late onset 2 l oOH deficiency is expressed in individuals with the genotype 21-OHS/21-OI:'P or in individuals who have one 21-OH s allele and one 21-OH" allele. Linkage of the 21-OH ° allele to HLA has been established by the earlier studies of congenital 21-OH-def [2-7]. Linkage of the 21-OH s allele to HLA cannot yet be established because of the limited number of families studied to date, but the following observations make this the
64
Z. Laron et al. most likel~ conclusion: The only HLA-identi~:al sibling to a patient in any of these five families has the same biochemical abnormality as the patient (family T); no other siblings of patients have significant biochemical abnormalities; the existence of alternative alleles at the same locus is the most likely explanation for effects on the activity of the same enzyme; and, finally, the individuals in this study who carry the 21-OH t allele, all carr~ it in association with the same HLA haplotype: B14,DRwl,BfS. The association of late onset 21d3H-deficiency with a particular HLA haplotype in the Ashkenazi population is another finding of this study. If it is assumed that the fathers in families G and L are homozygous for the B14,DRwl haplotype (see above), 8 out of 8 haplotypes in the probands and 10 out of 10 total haplotypes of Ashkenazi origin that are associated with 21-OH' alleles have the determinants BI4,DRwl and BfS. The average gene frequency for B14 in 9~4 Ashkenazi haplotypes of German, Polish, Russian, and Rumanian extraction can be calculated from the data published by Bonn6-Tamir et al. [19] as 0.118. B14 haplotypes are thus signiftcantly incrvased among the patients ( ~ =48.71) imd among the patients plus biochemically abnormal fathers (X~ = 61.82), and this is statistically signifgant even after correction for the number of antigens being tested. No comparative data is yet available regarding the frequency of DRwl or the B14,DRwl haplotype among control Ashkenazis. It is important to note that the syndrome is associated in the Ashkenazi population wi.-h an HLA haplotype, BI4,DRwI,BfS, rather than with a single HLA determinant. Five of the hapiotypes also have the A locus antigen Aw33. It is possible that this haplotype association is caused by a classical "founder effect." FI6wever, we have also found 21-OH s alleles in association with BI~I haplotypes in two non-Ashkenazi Caucasian families (unpublished observations), ,-nd the geographical and ethnic dispersion of the association suggest that such a "founder effect" would have to be of relatively ancient origin. Because of its association with a particular HLA haplotype, the biochemical abnormality in late onset 21-OH deficiency has a remarkable similarit3, to the autosomal recessive inheritance of deficiency for serum complement C2 which shows strong genetic linkage disequilibrium with the HLA haplotype A25,B18,Dw2,BfS [20,21 ]; Idiopathic bemochromotosis is another disease with incomplete penetrance of clinical symptoms in which a biochemical abnormality is associated with complete HLA haplotypes (A3,B7 or A3,B14) [22,23~ For all three diseases it is thus characteristic that the disease gene is in genetic disequilibrium with one or two HLA haplotypes rather than with one HLA determinant alone. An association was also recently found in congenital 21-OH-def, with the haplotype A3,BwZtT,DRwT,Bt'F (M.S. Pollack, in preparation). Although this disease occurs in association with a large number of other HLA haplotypes [2zt~ the BwZt7 associated disease may represent a distinct variant. This paper demonstrates that the 21-hydroxylase locus can have at least three forms: The normal allele (21-OHn), the classical 21-OH-def allele (21-OH °) and the presently described 21-OH susceptibility allele (21-OHS). The presence of nonrandom gametic association in ,~ariants of both late onse¢ and congenital 21-OH-def suggests that the alleles expressed at the 2 l-hydroxylase locus may relate to the HLA determinants at the functional or molecular level. The 21-OH locus could, therefore, be part of the HLA supergene. Proof of the recessive, monogenic natare of the late onset endocrinological abnormality requires further family studies.
Late Onset 2 l-Hydrox.vlase Deficiency and HLA
65
ACKNOWLEDGMENT This work was supported by the U.S. Publk Health Services, National Institutes of Health, under Grants NCI-CA 22507, CA 17404, CA 08748, CA 19267, EY-01616; by Grant Rl~ 47 from the General Clinical Research Centers Program of the Division of Research Resources, NIH; and by Grant HD 00072.
REFERENCES I. McKusick VA: Mendelian Inheritance in Man, 4th ed. Baltimore, Johns Hopkins Universit.v Press, 1975, p.403. 2. Dupoat B, Oberfield SE, Smithwick EM, Lee TD, Levine LS: Close genetic linkage between HLA and congenital adrenal h.vperplasia (2 l-hydroxylase deficiency). Lancet 2:1309, 1977. 3. Levine IS, Zachmann M, New MI, Prader A, Pollack MS, O'Neill GJ, Yang SY, Oberfield SE, Dupont B: Genetic mapping of ~he 21-hydrox~,lase deficienc,v gene within the HLA linkage group. New Engl J Med 299:911, 1978. 4. Price DA, Klouda PT, Harris R: HLA and congenital adrenal hyperplasia linkage confirmed. Lancet 1:930, 1978. 5. Weitkamp LR, Br.vson M, Bacon GE: HLA and congenital adrenal hyperplasia linkage confirmed. Lancet 1:931, 1978. 6. Puchoit V, Fitzsimmons JS, Gelsthorpe K, Pratt RF, Doughty TW: HLA m~d congenital adrenal h.vperplasia. Lancet 2:1046, 1978. 7. Grosse-Wilde H, Well J, Albert E, Schoiz, S, Bidlingmaier F, Sippel WG, Knorr D: Genetic linkage studies between congenital adrenal h~-perplasia and the HLA blood group s.vstem, lmraunogenetics 8:41, 1979. 8. Gutai Jp, Kowarski AA, Bigeon CJ: The detection of the heteroz,vgous carrier for congenital virilizing adrenal hyperplasia. J Pediatr 90:924, 1977. 9. Well J, Bidlingmaier F, Sippell WG, Butenandt O, Knorr D: Comparison of two tests for heteroz,vgosit~' in congenital adrenal h.vperplasia (CAI-I). Acta Endocrinologica 91:109, 1979. 10. Lorenzen F, Pang S, New MI, Dupont B, Pollack MS, Chow DM, Levine IS: Hormonal phenot~,pe and HLA-,get~ot,vpe in families of patients with congenital adrenal hyperplasia (2 l-hydrox),lase deficienc.v). Pediatr Res 13:1356, 1979. 11. Newmark S, Dluh,v R, Williams G, Pochi P, Rose L; Partial 11 and 21 h.vdroxylase deficiencies in hirsute women. AmJ Obsttyt Gynecol 127:594, 1977. 12. Zachmann M, Prader A: Unusual heteroz.vgotes of congenital adrenal h.vp,~rplasia due to 2 l-h.vdrox.vlase deficienc.v. Acta Endocrinol (Kbh) 87:557, 1978. 13. New M1, Lorenzen F, Pang S, Gunczler P, Dupont B, Levine LS: "'Acquired" adrenal h.vperplasia with 2 [-hydroxylase deficienc.v is not the same genetic disorder as congenital adrenal h.vperplasia. J Clin Endocrinol Metab 48:356, 1979. 14. Alper CA, Boenisch T, Watson L: Genetic polymorphism in human glycine-rich beta-glycoprotein.J Exp Med 135:68, 1972. 15. K~mpfJ, Bissbort S, Ritter H: Red ceil glyoxalase I (E.C:4.4.1.5): Formal genetics and linkage relations. Humangenetik 28:249, 1975. 16. Quazi QH, Hill JG: A modified method for the estimation of pregnanediol, pregnanetriol and seven common 17-ketosteroids in urine by gas liquid chromatography. Steroids 22:311, 1973.
66
Z.I.aron et al. 17. Pang S, Hotchkiss J, Drash AL, Levine I.S, New MI: Microfdter paper method for 17a-hydroxyprogesterone radioimmunoassay: Its application for rapid :;creening for congenital adrenal hyperplasia. J Clin Endocrinoi Metab 45:1003, 1977. 18. P~acholtV, Fitzsimn~onsJS, Reynolds MA, Gelsthorpe K: Location of the gene for 2 l-hydroxylase deficiency (abstr~ts). The European Society for Pediatric Endocrinology. Ulm, September 1979. 19. Bonn6-Tamir B, Bodmer JO, Bodmer WF, Pickbourue P, Brautbar C, Gazit B, Nero S, Zamir R: HLA polymorphism in Israel 9. An overall comparative analysi.q. Tissue Antigens 11:235, 1978. 20. F~aSM, Stern IL Kunkel HG, Dupont B, Hansen JA, Day NK, Good RA, Jersild C, F,3tino M: MLC-determinants and C2 deficiency: LD-Ta associated with C2 deftciency in four families. J Exp Med 142:495, 1975. 21. Raum D, Glass D, Carpenter CB, Aiper CA, Schur PH: The chromosomal order of genes controlling the maior histocompatibility complex, properdi,q factor B, and deficiency of the second component of complement. J Clin Invest 58:12,|0, 1976. 22. Beaumont C, Simon M, Fanchet R, Hespel J-P, Brissot P, Gener.et B. Bourel M: Serum ferritin as a possible marker of the hemochromatosis allele. N Engl J Med 2~ ~.169, 1979. 23. Cartwright GE, Edwards CQ, Kravitz K, Skolnick M, Amos DB, Johnson A, Buskiaer L: Hereditary hemochromatosis. Phenotypic expression of th~ disease. N EnglJ Med 301:175, 1979. 24. Pollack M, Levine L, Zachmann M, Prader A, New M, Oberfield S, Dupont B: Possible genetic linkage disequilibrium between HLA and the 21-hydroxylase deficiency gene (congenital adrenal hyperplasia). Transplant Proc 11:1315, 1979. 25. Tanner JM: Growth at Adolescence, 2nd ed. Blarkweli Scientific Publications, Oxf~,rd, 1962.