Tangier Disease (Familial High Density
   Lipoprotein Deficiency)"

Clinical and Genetic Features in Two Adults

HARRY N. HOPF~IAN, II, XD. and DONALD S. FREDRICKSON, M.D.

Rochester, Afinnesota

I N 1'961 Fredrickson and associates (7,2] de-
  scribed a new disorder of cholesterol and
liiloprotcin metabolism in two young siblings.
The significant clinical features were hypo-
cholcsterolemia and enlarged tonsils of unusual
appearance. One sibling also had hepatosple-
nomegaly and lymphadenopathy. Foam cells
observed in the tonsils and lymph nodes con-
tained large amounts of cholesterol esters. Both
siblings were found to have nearly complete
absence of plasma high-density or alpha1 lipo-
protein (HDL). In the absence of further knowl-
edge of the basic defect, the disorder was named
"Tangier disease," after Tangier Island, the
Chesapeake Bay home of the family. More re-
cently, two other cases in children of a family in
Missouri unrelated to the Tangier Island pa-
tients have been discovered [3,4].

Studies of the kindreds of these two involved
sibships revealed that many family members
had HDL concentrations well below those of
comparable control subjects [4]. The data ap-
peared to be consistent with the hypothesis that
control of plasma HDL concentrations resides
in a single pair of autosomal genes. The pheno-
type resulting from a single abnormal mutant is
characterized by low HDL concentrations; the
homozygous individual exhibits the full blown
syndrome of tissue storage of cholesterol esters
and has no significant plasma HDL.

The present report describes the fifth and
sixth cases of Tangier disease, which involve
two brothers. These are the first cases observed
among adults and provide new insight into the
manifestations and course of this hereditary

Bethesda, Maryland

disease. Included also are the results of plasma
lipid and lipoprotein determinations among
members of this kindred.

REPORT OF CASES

CASE 1. A forty-two year old, white, oil-refinery
worker who first registered at the Mayo Clinic in
hfarch 1962 was referred by his family physician for
the evaluation of splcnomcgaly. He had consulted his
physician in the fall of 1361 because of fatigue, at
which time mild anemia and splenomegaly were
found. The anemia was said to have diminished fol-
lowing iron therapy, but splcnomegaly and mild fa-
tigue persisted. The patient had been well otherwise and
able to work regularly; no other symptoms were elici-
ted except for intermittent mild diarrhea of several
years' duration, manifested as two to four soft to loose,
floating, occasionally foul-smelling stools daily. The
history disclosed none but the usual childhood illnesses.
He had undergone tonsillectomy at age fourteen in a
physician's of&x, but recalled little about prior
indications except recurrent sore throat and the
judgment that his tonsils were quite enlarged. A cyst
on the thyroglossal duct had been removed in 1945
while he was in naval service.

On physical examination, the patient appeared
generally healthy and alert. Weight was 172 pounds
and height 68 inches. Blood pressure was 135 mm.
Hg systolic and 90 diastolic. No skin lesions were
noted except for a few hyperpigmented areas over the
anterior tibia1 regions. The corneas had a slightly
cloudy appearance. Slit-lamp examination revealed
the clarity and relucence of the corneas to be defi-
nitely diminished, with random soft densities involv-
ing the entire stromal thickness of each cornea. In
the pharynx were several flat patches of yellow-orange
lymphoid tissue. No lymphadenopathy was noted.
The splenic tip was palpated 6 to 8 cm. below the left

  * From the Section of Medicine, Mayo Clinic and Mayo Foundation, Rochester, Minnesota, and the Section
on Molecular Diseases, Laboratory of Metabolism, National I Ieart Institute, Bethesda, Maryland. This investigation
was supported in part by Research Grant AM-06908 from the National Institutes of Health, Public Health Service,
to the Gastrointestinal Research Unit, Mayo Clinic and Mayo Foundation. Manuscript received October 26, 1964.

582      AMERICAN JOVRNAL OP MEDICINE


Tangier Disease-HoJman, Fredrickson               583

TABLE I

LABORATORY FINDINGS IN TANGIER DISEASE

Tests                               -

Ifemoglobin (gm./lOO ml. blood). .
Erytluocytes (million cu. mm. blood).
Leukocytes (cu. mm. blood).
Sedimentation (mm. hr., \\`estergren).
Rcticulocytes (per cent).
Bromsulfalein retention (per cent).
l'rotIuombin time (sec.). .
Iron (pg./100 ml. serum). .
Sugar (mg./lOO ml. blood).
Bilirubin (mg. / 100 ml. serum)
Direct...............................
Indirect
SCOT (rMlhour/ml.). . .
Phrxphatase

Alkaline (King-Armstrong units/100 ml.
serum). . . .
Acid

  l`otal (nessey-LowTey units). . .
  Tartrate-inllil)ita~le (King-Armstrong
   units/100 1111. serum).
Carotene (pg. 1100 ml. serum).
Calcium (mg./lOO ml. serum)
Uric acid (mg./lOO ml. serum). .
Protein (gm./lOO ml. serum)
All
   mmm..............................
Alpl~a, globulin.. _. .
hlplla, globulin..
Beta globulin.
Gamma globulin.. . .
Fecal nitrogen (gm./dayt). .
Fecal fat

gm.perdayt..........................
Per cent of feral solids t

II

-

Normal         Case 1         Case 2

  14-17          13.2           14.2
4.2-5.5          4.73          4.65
5,000-9,000     3,300        6,000
   O-20          10              
0.5-2.5         2.5, 10          
<6             8             0
  17-19          21            20
  50-l 75        120            126
  65-90          84            76

0             0             0
<0.6            1.5            1.94
<1.43           1.61           1.34

<14

17

5.9

0.13-0.63         0.45           0.34

co.7            0.07          0.09
>G3           26.9           27
8.9-10.1         10.0            9.6
3.8-7.1        9.1,9.6*      9.0,lO.l

3.3-4.3          4.27           4.28
0.3-0.4          0.48           0.36
0.5-0.8          0.5           0.43
0.6-1.1          0.71           0.93
0.8-1.6          0.94           1.21
<2.5            1.6           0.75

<7.5
>30

9.66
30

4
17

-

* By uricase method, 9.4 (courtesy of Dr. J. Seegmiller).
t 72-1101~ collection while on 100 gm. Sat diet.

costal margin, and the liver edge was noted 2 to 3 cm.
below the right costal margin.

General laboratory findings are given in Table I.
(Plasma lipids and lipoproteins are described in a
later section.) A smear of the peripheral blood showed
anisocytosis, polychromasia, mild leukopcnia and
thrombocytopenia. Biopsy revealed that the sternal
marrow was active, with many foci of foam cells sur-
rounded by excess of eosinophils. The chest roent-
genogram did not show any abnormality. The chole-
cystogram revealed a normally funcLioning gall-
bladder. Roentgenograms of the long bones and of the
stomach, small intestines and colon did not show any
dcrccts.

Proctoscopy (by Dr. R. J. Spencer) revealed the
rectal mucosa to be diffusely pale yellow and studded
with tiny flat, discrete, orange-brown spots through-

VOL. 39, OCTOBER 1965

Results

I -

out that portion of the bowel visualized. Biopsy of the
rectal mucosa showed large histiocytes filled with
foamy cytoplasm in the mucosa and submucosa.
Peroral biopsy of the jejunal mucosa, using the
Crosby-Kugler capsule, did not disclose any abnor-
malities. A diligent search was made for foam cells or
accumulated intracellular lipid in the biopsy speci-
men, but none was seen, not even with special stains.
A liver biopsy specimen showed one small intra-
lobular cluster of foam cells. Special lipid staining of
the biopsy specimens of the liver and rectal mucosa
(by Dr. A. H. Baggenstoss) showed that the foam
cells and some small granules in the hepatic paren-
chymal cells reacted positively with the oil red 0
stain and gave a faint positive reaction to the periodic
acid-Schiff stain. The Schultz reaction for cholesterol
showed small foci of blue-green coloration in the


584           Tangier Disease-Hogman, Fredrickson

FIG. 1.  Case 1. Spleen showing lipid-filled reticulo-
endothelial cells of pulp. Hematoxylin and eosin stain,
original magnification X 250.

parenchymal cells and brilliant blue-green staining
of the foam cells in the rectal tissue.

No abnormalities were noted (by Dr. J. A. Gregg)
in the relative proportions of the unconjugated bile
acids and their taurine and glycine conjugates in a
specimen of bile obtained by duodenal intubation
[5]; the total bile acid concentration was within the
lower range of normal. The free amino acids in a 24-
hour urine sample were normal, and the alpha-amino
nitrogen content was 210 mg. (as determined by Dr.
J. D. Jones). The serum contained normal amounts of
total and alpha1 and alpha2 globulin glycoprotein [G]
(as established by Dr. \V. F. McCuckin).
Since then, the patient has been re-evaluated at the
Mayo Clinic or National Institutes of Health every
three to six months. Plasma cholesterol values have

TABLE II

SPLEEN LIPIDS (MG./GM. WET TISSUE)

Data         Case 1        7

Total lipid..
Phospholipid.
Phosphatidyl choline,
Sphingomyelin.
Cholesterol

Free...............
Esterified
Cholesterol-ester fatty
acids (estimated).
Triglyceride.
Lipid hexose.

-(-

A

50.7    20.6
15.4    10.4
4.6     2.1
2.3     2.0

4.8
7.8

5.5
2.0
0.2

4.0
0.7

0.5
0.4
0.2

I

Controls *

B

-

* Control tissues were obtained from (A) a child
and from (B) an adult who had died following cardiac
surgery. Both tissues showed histologic evidence of
chronic passive congestion.

remained low. The spleen has increased in size, and
on each visit the patient has complained of increased
upper abdominal fullness and discomfort. Thrombo-
cytopenia and leukopenia have become more marked,
along with reticulocytosis and other evidence of in-
creased regeneration of erythrocytes, although hemo-
globin values have not decreased signilicantly.

In September 1963 the patient complained of dis-
abling physical discomfort produced by the spleen,
the inferior margin of which was then below the level
ol the umbilicus. The liver had not increased in size
since the initial examination. Splenectomy was per-
formed on September 7. At operation, the spleen was
found to be greatly enlarged and much firmer than
normal. It weighed 1,160 gm. and was yellowish red.
The liver was only slightly enlarged and appeared
normal grossly. There were many enlarged lymph
nodes along the entire root of the mescntery. The
other findings at intra-abdominal exploration were
within normal limits. Microscopic sections of the
spleen showed the reticuloendothclial cells of the pulp
to be filled with intracytoplasmic lipid droplets.
There were also scattered clusters of cholesterol crys-
tals in the pulp. (Fig. 1.) Biopsy of a lymph node
showed marked fatty change. The results from anal-
ysis oC spleen lipids are shown in Tables u and III. The
patient's convalescence was uneventful, and he re-
sumed a normally active life.

In February 1364 he noted enlargement of the
right breast. Other findings on physical examination
at this time were within normal limits. Excisional
biopsy of the right breast disclosed benign hyperplasia
of the ductile tissue and stroma. Staining for fat did
not reveal any foam cells in the resected tissue. Labo-
ratory studies on two occasions showed the alkaline
phosphatase to be 28 and 34 King-Armstrong units.
The bromsulfalein test showed 20 per cent dye reten-
tion. The plasma total cholesterol was 125 mg. per

TABLE III

FATTY ACIDS IN CHOLESTEROL ESTERS FROM SPLEENS

Fatty        Case 1

Acid `*

12:o
14:o
15:o
16:0
16:l
17:o
18:O
18:l
18:2
20:4
24:l

I -

(%)

1.5
5.7
4.9
21.8
13.7
0.6
4.1
40.0
5.1
3.6
1.9

-T

-

Control Bf
(%)

0.8
0.3
31 .l
4.2

13.b
21.9
19.6
9.0

. . .

* Number carbons in chain: number double bonds,
t Source as in Table U.

AMERICAN JOURNAL OF MEDICINE


Tangier Disease-Hoj'man, Fredrickson

100 ml., and on paper electrophoresis an increase in
low-density lipoprotein (LDL) concentration was
noted.

CASE 2.  A farmer and distillery worker, the forty-
eight year old brother of the first patient, was seen at
the Mayo Clinic in March 1963 after a preliminary
survey of the family led to the suspicion that he also
might have Tangier disease. His medical history in-
cluded tonsillectomy in 1950 because of recurrent
sore throat and "enlarged tonsils." (The surgical
transcripts were obtained from his local hospital, but
there was no comment about the unusual features of
the tonsils and no histologic studies were reported.)
Typical angina pectoris of moderate severity had been
noted daily for the past five years. A prolonged epi-
sode of chest pain had led to the patient's hospitaliza-
tion near his home in 1961, but "no permanent dam-
age" had been noted. Dull, nonspecific, right abdomi-
nal discomfort of several years' duration was the pa-
tient's only other current complaint. No history of
diarrhea or steatorrhea could be elicited.

On physical examination, the weight was 208
pounds and height 67 inches. The blood pressure was
142/100 mm. Hg. The only skin lesions were two 3
mm. yellow-brown spots on the lower calf and tibia1
areas. There was no cornea1 clouding on gross exami-
nation, but slit-lamp examination showed a hazily
flocculent infiltration throughout the cornea1 stroma.
This was described by an ophthalmologist observer
(Dr. Ludwig Von Sallmann) as "decreased trans-
parency of the cornea, more marked centrally than
peripherally, and more prominent in the posterior
than the anterior stroma. This slight diffused opacity
can be resclved into fine equidistant dots." Fundu-
scopic examination revealed minimal sclerosis of the
retinal arterioles in both eyes and a fluffy retinitic
spot in the inferior temporal region of the left. Cystic
limbal lesions thought to be pingueculae were present
bilaterally. In the pharynx a few follicles remained,
surrounded by a pale yellow-orange ring in several
instances. The liver edge was palpable at the right
costal margin ; the spleen was not palpable. The other
physical findings were not remarkable.

General laboratory findings are listed in Table I.
The standard thoracic roentgenogram, roentgeno-
grams of the gallbladder, colon and lumbar vertebrae
and the excretory urograms did not reveal any ab-
normality. An electrocardiogram showed a non-
specific repolarization abnormality in the mid-pre-
cordial leads. Marrow aspirated from the iliac crest
contained a few macrophages ("foam cells"), their
cytoplasm loaded with doubly refractile droplets.
Many other scattered droplets appeared to be out-
side any cytoplasmic membrane. The remaining
marrow elements were normal. Proctoscopic examina-
tion revealed a smooth yellowish mucosa with tiny,
flat, dark red-brown 1 mm. spots scattered through-
out. A rectal biopsy specimen showed mucosal and

VOL.  39, OCTOBER 1965

FIG. 2.  Case 2. Rectal biopsy specimen showing large
lipid-laden cells in lamina propria and beneath mus-
cularis mucosa. Hernatosylin and eosin stain, original
magniIicalion X 150.

submucosal collections of foam cells. Biopsy of tonsil
and adenoid remnants revealed clusters of pale-stain-
ing foamy macrophages. A jejunal biopsy specimen
did not show any histologic abnormalities, but a needle
biopsy specimen of the liver revealed one small intra-
lobular focus of foam cells, as in the previous case.
The foam cells from pharyngeal, rectal and liver
biopsy specimens exhibited histochemical character-
istics identical with those in Case 1. (Fig. 2.)

The patient's symptomatic coronary heart disease
was discussed with him, and he was also advised to
reduce his weight. On July 22, 1963, he died suddenly
while driving his farm truck. Necropsy was not
performed.

METHODS

Measurements of cholesterol [7], phospholipid (S]
and triglycerides [9] were performed on chloroform-
methanol extracts [7U] of tissue, plasma or lipoprotein
fractions isolated by preparative ultracentrifugation
[77]. Tissue was homogenized in chloroform: meth-
anol (2: l), using two 10 to 20 ml. portions of solvent
per gram of wet tissue. After the combined extracts
were filtered and the chloroform phase washed ac-
cording to the method of Folch and associates [72],
they were kept at 4"~. until analyzed. Total lipid was
determined gravimetrically and lipid hexose was
measured by the anthrone method [73] following


586           Tangier Disease-Hofman, Fredrickson

      TABLE IV
PLASMA LIPlDS (MC./100 ML.)

                       Cholesterol           ,

    source                                   I    Phospholipids       Triglycerides
                    Total        /    Free
            -__     I I
   Cax 1         38 (30-45)           14        86 (68-104)       142(136-152)
   Case 2         69 (50-88)           21        114(104-124)       213(195-238)
  Controls ;    250 (176-324) : I
                                     .       280 (205-355)       70 (20-120)

NOTE: Values for phosphohpids and triglycerides of patients represent two to five postabsorptive samples obtained
on different days. Control cholesterol and phospholipid values represent mean (and 90 per cent confidence limits)
obtained from forty-three normal men forty to forty-nine years old, averaging 44.5 years of age. Control triglyceride
values were derived from major peak of distribution of values obtained from 390 fasting and apparently healthy
subjects [23].

           TABLE v
ULTRACENTRIFUGAL ANALYSIS OF LIPOPROTEINS
     (MC./100 ML. PLASMA)

Lipoprotein
Fraction

   Case 1               Case 2               Controls *

ct      =t         C         PL         C          PL

Plasma
<1.019
1.021-1.063
1,063-l .21
>1.21

    30        68         50        104        171         215
    19         44         33         70         26         31
    11         15          13         23         97          69
     1   1 1 :: / :       43     111
i O                                         0         21

* Mean of samples from ten men, aged twenty-one to twenty-eight years. From Have1 et al. [ 7 I]
t C = cholesterol. PL = phospholipid.

hydrolysis of the sample at 100'~. for 15 hours in 6 N
hydrochloride. Phospholipids were separated and
quantified by the method of Skidmore and Entenman
[ 74) using two-dimensional thin layer chromatog-
raphy. Cholesterol esters were isolated by chroma-
tography on alumina. Methyl esters of fatty acids
were chromatographed on 15 per cent ethylene glycol
succinate at 195'~. The ionization detector was
calibrated for response with NIH Metabolism Study
Section standards. Paper electrophoresis for lipopro-
teins was performed by the technics of Swahn [75]
and of Lees and Ilatch [/G].

RESULTS

Plasma Lipids and Lipoproteins. In the tlvo
cases, the plasma lipids (Table IV) followed the
abnormal pattern which is characteristic of
Tangier disease, although not necessarilyunique.
This is the association of low levels of cholesterol
and phospholipids with normal or elevated con-
centrations of glycerides. On the basis of the
known composition of the major plasma lipo-
protein systems, it may be predicted from this

lipid pattern that there \vill be a deficiency in
HDL or in that portion of LDL of density be-
tween 1.019 and 1.063, with an associated fmd-
ing of normal or elevated concentrations of tri-
glyceride-rich LDL of density less than 1.019.

In these cases all three features proved to be
present. On paper electrophoresis, no alphai
lipoprotein could be seen. On preparative ultra-
centrifugation (Table v), there was no significant
HDL in the appropriate density band, 1.063 to
1.21. Also, there was a definite decrease in LDL
of density 1.019 to 1.063 (which corresponds to
beta lipoproteins on paper electrophoresis or Sf
0 to 12 in the analytical ultracentrifuge) and
probably a significant increase in LDL of den-
sity less than 1.019 in Case 2.

Immunoelectrophoresis on agar, using either
rabbit antibodies to human HDL or horse anti-
human serum, revealed no precipitation line
attributable to HDL. The results of agar diffu-
sion studies are shown in Figure 3A. The rabbit
anti-HDL serum used in these experiments has

AMERICAN JOURNAL OF MEDICINE


Tangier Disease--lZ~J~nan, Fredrickson

587

FIG. 3.  A, precipitation lines formed by Ouchterlony agar dillusion technic with serum from normal subject (I ),
Cav 1 (2), Case 2 (3) and patient with acanthocytosis and abetalipoproteinemia (4) against rabbit antibodies pre-
pared against human lipoproteins of density 1.063 to 1.21 ("anti-l IDL"). Both precipitation lines at (1) are attributed
to HDL (see text). H, arrangement same as in A, except rabbit antibodies were prepared against human lipoprotein
of density 1.063 ("anti-LDL"). Here both patients with Tangier disease demonstrated LDL, which was further shown
by immunoelectrophoresis.  ThFse plates were prepared by Dr. Robert I. Levy.

been shown to detect two different HDL anti-
gens and to contain no antibodies to either
albumin or LDL [ 171. A very faint precipitation
line was formed by serums from both patients,
suggesting the presence of a very small amount
of HDL. (Fig. 3A.) This is very similar to results
obtained in the four other known cases of Tan-
gier disease, and precise identification of the
faintly detectable antigen is under way. In
contrast to serum from a patient with abetalipo-
proteinemia (kindly supplied by Dr. Leonard
Laster), serums from both of the present pa-
tients had easily detectable LDL, antigenically
similar to that in normal subjects although some-
what decreased in amount. (Fig. 3B.)

The abnormal concentrations in the LDL
fractions were further associated with lower than
normal cholesterol : phospholipid ratios in these
lipoproteins. Both of these abnormalities have
also been observed in lesser degree in the four
children with Tangier disease [IS].

Tissue Lipids. The significant splenic abnor-
mality in Case 1 (Table II) was the presence of
cholesterol esters in amounts which far exceeded
that in the two control tissues analyzed concur-
rently. The fatty acid pattern of the cholesterol
esters in the spleen in this case (Table III) was

VOL.. 39, OCTOBER 1965

similar to that seen earlier in tonsils and lymph
nodes from the children with Tangier disease
[Z]. Oleic acid predominated and the relative
amounts of linoleic and palmitic acids were less
than those seen in the control spleen. (Table III.)

A considerable amount-about 30 per cent-
of the total lipid of the spleen in Case 1, as esti-
mated gravimetrically, could not be accounted
for in the summation of the cholesterol, phos-
pholipid and glyceride fractions. (Table II.)
Such measurements were made a total of nine
times on three different extracts of the abnormal
spleen. Since 13 to 22 per cent of lipid was un-
accounted for in the controls also, it is likely that
the gravimetric measurements of total lipid were
uniformly and inexplicably too high; but the
possibility of an abnormal and undetected lipid
in the tissue in Case 1 has not been excluded
completely.

The lipid hexose content was normal, how-
ever; and the total fatty acid content, deter-
mined by quantitative gas chromatography
after  saponification  and  methylation,   was
equivalent to that predicted from the quantities
of estcrified lipids specifically measured and pre-
sented in Table II. The gas chromatographic
pattern of the total fatty acids in the spleen


588

Tangier Disease-I-logman, Fredrickson

       O Normal HDL    O Absenl HDL
       GLOW HDL         t Died

        All those ~n generotion I.lI.IUW!Z-deceased

FIG. 4.  Pedigree of Kentucky propositi, showing distribution of HDL ccncentratiom among forty-eight relatives.
See Table VI for details of HDL values.

differed significantly from that of control tissue
(spleen B, Table III) only in an increased per-
centage of oleic acid, attributable to the in-
creased amount of this acid esterified with
cholesterol. Within the limits of detection of the
methods employed, the abnormality in lipids
from the spleen in Case 1 was qualitatively the
same as that previously found in other tissues
from children with Tangier disease [2,78].

STUDY OF THE KINDRED

The patients described herein are two of six
children born to parents of seemingly pure
English ancestry, both of whose families have
lived in Kentucky for several generations. Two
female siblings are living. (~1-23, ~1-30, Fig. 4
and Table VI.) A male and a female sibling each
died at about two years of age from "throat

trouble" said possibly to have caused them "to
choke to death." The two surviving sisters of the
patients appear to be in good health. The tonsils
of one have been removed but no records con-
cerning them are available. The tonsils of the
other appear normal. Their mother (v-34, Fig.
4 and Table VI) is seventy-nine years of age and
in good health. The patients' father (v-30, Fig. 4
and 5) died of a myocardial infarction at the
age of seventy-five.

Most of the kindred reside in a small area of
western Kentucky, into which their forebears
migrated in the late eighteenth and early nine-
teenth centuries. Nearly all their contemporary
associates likewise were of Anglo-Saxon ancestry
and crossed the Appalachians from the eastern
seaboard states. Antecedents of the present
kindred have been traced to Pennsylvania,

AMERICAN JOURNAL OF MEDICINE


v-2
v-3
v-4
v-9
v-17
v-21
v-24

v-26
v-27
v-28
v-29
v-32
v-34
v-37
V-41
VI-4
VI-9
VI-1 0
VI-1 I
w-1 2
m-1 3
VI-1 6
VI-17
VI-1 9
VI-20
VI-2 1
VI-22
w-23
~1-25
~1-26
w-27
~1-28
VI-30
VI-31
m-35
~1-36
~1-38
VI-39
VI-40
VII-5
VII-7
VII-1 2
WI-1 3
w-1 4
VII-1 5
VII-1 6
w-1 7
WI-1 8

Tangier Disease--Ho$man, Fredrickson              589

DISTRIBUTION OF LIPID FRACTIONS AMONG KENTUCKY KINDRED

Age (y-r.1
and Sex

79, F
77, F
75, F
64, M
71, M
85, F
79, M
75, M
56, F
69, M
68, M
68, M
79, F
87, F
73, M
36, M
56, F
57, F
55, F
48, F
42, F
49, M
43, M
35, M
46, F
45, F
36, M
58, F
45, F
48, M
44, M
43, F
36, F
34, M
36, M
37, M
60, M
69, M
65, F
19, M
10, F
19, F
10, M
19, M
15, M
13, M
35, F

25, F

TC*

280        297
299        347
344        376
294        331
174        250
167        218
261        273
181        242
221        313

Ii;;     2%
373        353
160        190
118        166
205        248
229        301
213        305
418        389
245        315
250        239
284        304
232        281
174        274
234      .316
215        266
132        176
157        222
278        347
241        298
88        115
36        104
199        220
115        159
228        233
252        317
306        258
226        243
236        287
241        331
136        187
168        200
138        151
102        170
105        151
108        203
107        196
154        194
174        228

-

--

-

Plasma            Density >l ,063

PL'       TG*

iii
is5
148
209
215

ii;
iii
116
&ii
178

`Ii,
174
213
104
ii;

ii6
138
49
54

iii

`ii
t31
41
79
76

-

-

TC

66
50
72
64
33
22
34
24
49
15
19
53
30
20
52
30
53
79
59
81
58
117
34
37
29
33
29
44
30
1
1
52
35
35
44
53
39
31
58
36
45
29
29
35
36
32
59
61

PL

HDL
Pheno-
typic
Classifica-
tion?

144        N
128        N
170        N
158        N
97        N
74        L
94        N
82        L
144        N
72       -L
75        L
122        N
67        L
67        L
136        N
91        L
138        N
184        N
161        N
171        N
131        N
160        N
115        N
107        N
93        L
93        L
87        L
121        N
101        L
14        A
8        A
111        N
89        N
85        N
134        N
104        N
107        N
100        L
148        N
102        N
131        N
70        L
96        L
88        N
108        N
105        L
136        N
150        N

* TC = total cholesterol. PL = phospholipids. TG = triglycerides.
t N = normal HDL. L = abnormally low. HDL (heterozygous). A = absent HDL (homozygous).

VOL. 39,  OCTOBER 1965


Tangier Disease---Hoffman, Fredrickson

              cl Normol vision
            O Retinitis pigmentoso
Fro. 5.  Pedigree of Kentucky prppositi, showing distribution of retinitis pigmentosa. Numbers are same as

in Figure 4. -

Virginia and the Carolinas. However, there
were no names common to the population of
Tangier Island nor were any traceable connec-
tions between the affected Kentucky, Missouri
and Tangier kindreds discovered.

Blood samples were obtained in the nonfasting
state for analysis of plasma HDL, as previously
described [#I. The same criteria were employed
for assignment of phenotype of HDL concentra-
tion as had been utilized in the study of the
other two kindreds. HDL was quantified by the
amount of cholesterol obtained in the prepara-
tive ultracentrifuge fraction of density greater
than 1.063; and "abnormally low" values were
defined as those below the lower 5 per cent of
control values, or 33 and 35 mg. per 100 ml. in
male and female subjects, respectively [4].

The distribution of HDL concentrations in
the kindred is shown in Table VI and Figure 4.
The mother of the propositi has a deficiency of
HDL, as do relatives of both of the parents, who
were second cousins. Judging by the number of
his abnormal siblings, the father of the propositi
was probably abnormal-a circumstance in
keeping with the recessive mode postulated for
Tangier disease from previous studies. In Figure
4 are two phenotypes inconsistent with the
hypothesis that a single abnormal gene should
cause deficiency of HDL. One (VII-~ 6) is a hetero-
zygote whose parents are normal. More impor-
tantly, the other (VII-14), the son of the patient
in Case 1 (~1-27) who should be heterozygous
abnormal, appears to be phenotypically normal.

The HDL concentrations in both these subjects
(Table VI) vary from the arbitrary limit of normal
by only 2 mg. per 100 ml., which is within the 10
per cent experimental error of the methods [4].

The patient in Case 2 and his wife (~1-25) had
only two children (~11-12, ~11-13) both of whom
have low plasma concentrations of HDL. There
is no history of stillbirths or miscarriages to sug-
gest less than normal viability of a homozygous
abnormal offspring theoretically possible from
this mating.

With allowance for the uncertainty in detect-
ing the heterozygous phenotype, the data in the
present pedigree indicate a familial abnormality
in maintaining plasma HDL concentrations
which is consistent with the genetic mode earlier
postulated for Tangier disease [4].

RETINITIS PICMENTOSA

A number of living family members have
marked visual impairment. In every instance
night blindness was first noted in adolescence or
early adulthood; in most instances it progressed
to near blindness. Several ophthalmologists who
examined a number of the affected persons have
described this disorder, commonly known among
family members as "the moon eye," as retinitis
pigmentosa.

The distribution of retinitis pigmentosa is
shown in Figure 5. The retinopathy is associated
with descendants of 111-15. Seven of these descen-
dants with retinitis were not available for blood
sampling and are not included in the pedigree

AMERICAN JOURNAL OF MEDICINE


Tangier Disease--NoJman, Fredrickson

591

chart. (Fig. 4.) Among those persons in whom   gross screening technic, but some of the usual
the diagnosis of retinitis pigmentosa seems  staining technics define alphai lipoprotein (the
reasonably substantiated, the concentration of  electrophoretic equivalent of HDL) less well
HDL is low in five and normal in one. It should   than other lipoproteins and may falsely indicate
be noted that both patients described herein   absence of HDL when actually the concentra-
were examined on several occasions by compe-   tion is just somewhat lower than normal. Starch-
tent ophthalmologists, and no evidence of  block electrophoresis is better for defining alphai
retinitis was seen. The data suggest that the  lipoprotein because larger amounts of plasma
defect in HDL and the retinitis pigmentosa are  can be loaded, providing a deeper color with
not linked in this kindred.             lipid stains.

COMMENTS

There remains little doubt that the two adults
described herein have the same genetically
determined disorder described previously in
children as Tangier disease. Each has a defi-
ciency of plasma HDL quantitatively as marked
as that in the children, persistent tags of abnor-
mal-appearing tonsil tissue and foam cells in
many tissues without evidence of accompanying
granulomatous or other inflammatory changes.
Excessive storage of cholesterol esters in the
spleen of one patient has been demonstrated.
The fatty acid moiety of these esters is also the
same as in other cases, although it has not been
proved that this is a useful basis for distinguish-
ing the biochemical defect. Finally, many rela-
tives have abnormally low plasma concentra-
tions of HDL, the distribution being comparable
to those found in the two pedigrees of the child-
hood cases [4].

The discovery of two more cases of Tangier
disease is noteworthy for several reasons. It
suggests that the condition may not be extremely
rare and that physicians will find it worthwhile
to keep in mind the simple clues to its diagnosis.
These are hypocholesterolemia in the absence of
malabsorption and in association with foam cell
infiltration of tissues. A unique manifestation of
the latter appears to be an orange or yellow-
gray appearance of the tonsils or the remaining
mucosa after these usually enlarged tissues have
been removed. Thus the observation of unusual
tonsils or the presence in the oropharynx of one
or more follicles surrounded by a distinctive
light orange or yellow-gray ring is an indication
for determining the plasma cholesterol. This
should also be carried out in any instance of un-
explained enlargement of the liver, spleen or
lymph nodes, of cornea1 infiltration or of abnor-
mal appearing rectal mucosa.

If the cholesterol concentration is less than
125 mg. per 100 ml., HDL concentrations should
be determined. Paper electrophoresis is a useful

Preparative ultracentrifugation provides ac-
curate quantitation of HDL. However, the
definition of a lipoprotein in the ultracentrifuge
is an operational one, depending upon the ap-
propriate combination of lipid and protein to
impart a specific density to the molecules. There
are instances, as in severe hyperglyceridemia
[19] or obstructive liver disease [ZO], in which
deficiency of HDL or even apparent absence
may be secondary to a change in density or
physical state of the lipoprotein rather than to
an absolute decrease in quantity. Confirmatory
diagnosis of severe HDL deficiency, therefore,
depends upon immunochemical methods for
identification of the lipoprotein. Pure antibodies
to human HDL are not always readily avail-
able; but various anti-human serums are, and
these may be used in immunoelectrophoresis with
appropriate stains to identify the lipoproteins.
The present cases offer the first opportunity
to gauge the long-term effects of a presumably
congenital absence of significant circulating
HDL. These adults have lived four times as long
as the previously known patients. They demon-
strate that while Tangier disease is a relatively
benign process, compatible with reasonably
normal health at least to the fifth decade, it can
be associated with significant disability. The
presenting clinical problem in Case 1 was sple-
nomegaly. Initially the peripheral blood showed
mild leukopenia with a normal differential,
thrombocytopenia and increased red cell regen-
eration. These factors in conjunction with active
marrow suggested mild hypersplenism. The
hematologic findings became more abnormal
and, together with progressive splenomegaly,
led ultimately to splenectomy. Severe coronary
heart disease was present in Case 2 without
splenomegaly or hematologic abnormalities.
Any influence of disturbed cholesterol transport
and storage in the pathogenesis of this fatal dis-
ease is presently unknown. One child with
Tangier disease (Case 3) has a congenital pul-
manic stenosis [3].

VOL.  39, OCTOBER 1965


592

Tangier Disease--H@man, Fredrickson

Hitherto unappreciated sites of lipid storage in
Tangier disease were demonstrated in the gut
and eyes of the two adult patients. Gastrointes-
tinal symptoms (mild intermittent diarrhea) in
Case 1 led to proctoscopic examination, which
revealed a unique gross appearance of the rectal
mucosa in association with mucosal and sub-
mucosal foam cell infiltration. Identical findings
were present in Case 2. The suggestively steator-
rhea1 nature of the stools in Case 1 was investi-
gated by an intake-excretion study of fecal fat
and nitrogen. The value for fecal fat was slightly
in excess of normal. (Table I.) Both of the
brothers also had low serum concentrations of
carotene. However, the absence of other evi-
dence of malabsorption, despite roentgenog-
raphy of the small intestine and biopsy of the
jejunal mucosa in Case 1, militates against a
primary or secondary malabsorption syndrome
which might be responsible for the abnormal
serum lipid and lipoprotein findings in these pa-
tients. Moreover, in Case 2 the patient had no
gastrointestinal symptoms and was obese, and
his fecal fat and nitrogen excretions were normal.
No foam cells or other abnormalities were seen
in the biopsy specimens of jejunal mucosa from
either patient. Gut biopsies have not been per-
formed in the four children with Tangier disease;
but they, too, have had no evidence of gastro-
intestinal dysfunction.

The cornea1 clouding and stromal deposits
observed on slit-lamp examination presumably
are due to deposition of cholesterol esters in a
manner similar to that occurring in other tissues.
Moderate hyperuricemia without evidence of ab-
normal renal function was present in both cases,
but its significance here cannot be determined.

The basic inheritable defect in Tangier dis-
ease remains to be established with certainty.
HDL is one of the major mechanisms for trans-
porting lipid in extracellular fluid, others being
the albumin-free fatty acid complex and LDL
[21]. From a physiologic standpoint, particulate
emulsions such as chylomicrons may be con-
sidered a fourth mechanism, although their
chemical and functional relationships to the
other lipoproteins are not fully understood. One
of the major contributions of studies of Tangier
disease has been to point out an apparent lack of
absolute requirement for HDL in the absorption
of fat and formation of chylomicrons [78].

The polypeptide in HDL is specific and dis-
tinct from that in LDL. From the data available
it seems most likely that inability to maintain

significant concentration of plasma HDL in
Tangier disease is due to a genetically deter-
mined defect in elaboration of this polypeptide.
If this be true, the widespread accumulation of
cholesterol esters in tissues suggests that HDL
may have some specific function in maintaining
the equilibrium of cholesterol between plasma
and tissues.

It is noteworthy that there are also abnor-
malities in LDL in Tangier disease. In the adults
they have been especially marked (Table v), but
qualitatively similar findings have been present
in the affected children [18]. The hypocholes-
terolemia in Tangier disease thus is not solely
due to an absence of significant HDL, although
the latter lipoprotein class is by far the most
profoundly affected. In the ultimate explanation
for Tangier disease as based on alteration at a
single gene locus, it will be necessary to account
for changes in all classes of lipoproteins as, at the
least, secondary phenomena.

One of the few conditions in which cholesterol
esters are stored selectively in reticuloendothelial
tissues is eosinophilic granuloma [22]. Neither
the diagnostic granulomatous changes nor other
typical clinical features are found in Tangier
disease. One of us (D. S. F.) has had oppor-
tunity to measure plasma HDL in two patients
with an active form of the disseminated variety
of eosinophilic granuloma. In one, HDL was
normal; in the other, it was abnormally low
(HDL cholesterol measured 24 mg. per 100 ml.,
the mean for her age and sex being 54). How-
ever, the decrease was not comparable to the
near absence seen in Tangier disease [23].
Eosinophilic granuloma is not a familial disease,
and tonsillar abnormalities have not been re-
ported in association with it.

While the frequency of the gene or genes asso-
ciated with Tangier disease must undoubtedly
be quite low in the general population, the
finding of six cases within a three year period of
awareness of the disease suggests that more are
to be found. It still is less common than its
counterpart abnormality, abetalipoproteinemia
[24,23'3. The latter is characterized by the
absence of both the LDL and particulate forms
of lipoproteins and is associated with acantho-
cytosis,  steatorrhea, neurologic changes and
retinopathy. While LDL was reported as being
deficient in the parents of the patient first de-
scribed [2J], no definite mode of genetic in-
fluence on the development of this latter disease
has yet been established. The extension of clini-


Tangier Disease-HoJman, Fredrickson

cal observations in Tangier disease to adults
suggests that severe deficiency of HDL is asso-
ciated with a more benign long-term course than
abetalipoproteinemia. Both of these diseases
offer unusual opportunities for further exploring
the functions and control of the major plasma
lipoprotein systems, and all new patients deserve
careful study and extensive documentation.

SUMMARY

Two brothers, aged forty-five and forty-eight,
with familial deficiency of high-density lipo-
protein (HDL) (Tangier disease) are described.
These are the fifth and sixth known patients and
the first adults with the disease. They demon-
strated the hypocholesterolemia, abnormal ton-
sils and tissue storage of cholesterol esters char-
acteristic of this condition. One brother had
hypersplenism and both had unusual infiltration
of the corneas and rectal mucosa, new findings
presumably related to the chronic course of
Tangier disease. One brother had coronary
artery disease which apparently caused his
death. Low plasma concentrations of high-
density lipoprotein were distributed among
family members in a manner similar to that
reported in the two other involved kindreds.

Acknowledgment: We thank Dr. Charles F.
Stroebel and Dr. Haddon M. Carryer of
the Mayo Clinic for their clinical contributions
to the management of these patients.

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American Journal of Medicine

volume 39. number 4, pages 582-593. copyright 1965 and printed in the U.S.A.
Published by Thr Reuben H. Dnnnelley Corpnrnlion, 466 Lexington Ave., New York 10017.