Tissue Antigens (1978), 12, 357-366 Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission from the author(s)
HLA-Determination in Families with Hereditary Ataxia Erna Moller, Bengt Hindfelt and * Jan-Edvin Olsson ' Department of Clinical Immunology, Karolinska Institute, Huddinge Hospital, Huddinge and ' Department of Neurology, University Hospital, Lund, Sweden
In three families with hereditary ataxia, where the inheritance pattern was autosomal and dominant, HLA antigens were determined in 25 members. In two of the families, HLA linkage of disease was suggested, whereas in the third family, the data did not directly support this concept, since two recornbinational events between the postulated locus for disease and t h e HLA region had t o be assumed. However, with this assumption, our data are compatible with those of one family described recently (Jackson e t al. 1977) implying the presence o n the sixth chromosome, outside the HLA region, of a locus that determines the development of spino cerebellar ataxia (SCA). Further tests with definition of enzyme markers will have to be performed before conclusions as t o HLA linkage of a postulated SCA gene can be made. Received for publication 6 April, revised, accepted 2 June 1978
Several neurological disorders with uncertain mode of inheritance are associated with genetic markers of the HLA system, This is the case for multiple sclerosis (MS) (Bertrams & Kuvert 1972, Jersild e t al. 1972, Naito e t al. 1972) and myasthenia gravis (MG) (Pirskanen et al. 1972, Behan et al. 1973). Certain neurological diseases, e.g. the hereditary ataxias, have a pattern of inheritance which can be classified according t o clinical observations. However, even with a dominant inheritance, it is impossible t o predict which members of the family will subsequently develop clinical symptoms. In most cases this happens when the patients have grown up and already have children themselves. Since there is no treatment for these disorders it is essential to give genetic information and thereby prevent transmittance of the
disease. According t o Jackson et al. (1977) an ataxia locus is connected with the HLA locus on the sixth chromosome. The aim of our study was to prove whether or not determination of the HLA antigens is of clinical-genetic value in families with hereditary ataxias, where the circumstances indicate a dominant inheritance. Material Patients HLA antigens were determined in 25 members of three families with spinocerebellar ataxia(SCA) where the inheritance pattern was autosomal and dominant. In the first family (Fig. 1) several members were affected with a disorder consisting of a peculiar lower back and abdominal pain, defects of micturation and severe ataxia. Three affected members were examined.
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The onset of the disorder was usually between the ages of 20 and 3 5 . The initial symptom was a severe low back pain, radiating over the lower abdomen and down the inquinal regions. Concurrently, urinary urgency was conspicious. The symptoms were initially interpreted as being due t o urinary infections and treated accordingly without success. Subsequently the pain became sustained and incapacitating. Difficulties in emptying the bladder gradually evolved, necessitating the continuous use of an indwelling catheter. Within a few years, a progressive limbataxia appeared, predominantly in the legs. The affected members have all been of a slender body constitution. Somatic examination has been unremarkable, with the exceptions mentioned above. On neurological examination limb ataxia and a wide based gait disturbance were the dominating features. Cranial nerves were intact, apart from a slurred speech and some horizontal nystagmus. Intermittently, head tremor was observed. There were no pareses, although muscle mass seemed reduced. Stretch reflexes could not be elicited and plantar responses were flexor. Appreciation of touch, pain and temperature was normal, while vibration and positional senses were severely defective. As in other reported families, extensive laboratory tests on blood and cerebrospinal
0
A
Figure 1. Pedigrees of two families with autosomal dominant spinocerebellar ataxia. Left family I, right family 11.
fluid were non-contributory in elucidating the underlying pathogenesis. Myelographic studies did not reveal any morphological explanation t o the intense pain experienced early in the course. Despite normal neuroophthalmological findings, visual evoked responses revealed delayed lathencies (160-180 ms). Electromyography was consistent with a predominantly sensory neuropathy, leaving very few myelinated fibers intact. During the period of investigation, one of the affected siblings--born 1915-in the second generation died. At autopsy classical pathological features were found with demyelination of the dorsal spinal columns and the dorsal spinal ganglions. Degenerative changes were observed within the dentate nucleus, the Purkinje cells and the frontal cerebral cortex. The lumbo-sacral nerve roots were considerably smaller than normal, surrounded by fibrous tissue and exhibiting inflammatory infiltration. The full neurological syndrome affecting members of the second family (Fig. 1) was almost identical to that of the third family (Fig. 2), but the families were unrelated as far as could be traced. The earliest manifestations were observed at about the same age (20 to 40 years) and consisted of slurred speech and a progressive, ataxic gait disturbance. The temporal profile of neurological deterioration was also similar
HLA-DETERMINATION IN HEREDITARY ATAXIA
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4
Figure 2. Pedigree of one family (111) with autosomal dominant spinocerebellar ataxia.
I
111
6-40
4-45
6-41
A2 1 81218
A2 9 812140
09 NORMAL W 0
0
MALE FEMALE AFFECTED MALE, FEMALE DECEASED
Figure 3. Pedigree showing the HLA haplotypes in family 1. HLA-A and -B antigens are shown in haplotype form separated with vertical lines.
7-
I
5
0 NORMAL 0
MALE. FEMALE AFFECTED MALE, FEMALE DECEASED
Figure 4. Pedigree showing the tentative HLA haplotypes in family 11. HLA-A, - B and are indicated as in Fig. 3 .
-D
antigens
MOLLER ET AL.
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I
I
I
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?
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AFFECTED MALE. FEMALE DECEASED DECEASED AS CHILDREN NOT INVESTIGATED
Figure 5. Pedigree showing the HLA haplotypes in family 111.
in the two families. Medical examination was unrevealing, except for a tendency in some members of the family to develop moderate arterial hypertension. The neurological findings were identical in all respects to those encountered in the third family. Two examined members of the family (man born 1916 and woman born 1921, Fig. 4) presented the complete syndrome, while one male - borne 1954 -in the third generation (Fig. 4)exhibited ataxia on finger-nose and knee-heel testing. Another two male relatives in the third generation - born 1940 and 1949 (Fig. 4)-had somewhat equivocal findings, i.e. minor ataxia or tremor, weakened or absent Achilles reflexes. These subtle findings may indicate that they too are affected and perhaps will develop more clear-cut defects with time. Extensive laboratory tests on blood and cerebrospinal fluid in the affected members have not provided any clue to the underlying metabolic abnormality. Pneumoencephalogram in the man born 1916 has revealed a widened major cistern and an enlarged fourth ventricle, indicating an atrophic process. In the third family (Fig.
2) a neurological syndrome with features of Friedreich ataxia and sensory polyneuropathy has affected three females, presently alive and examined (Fig. 5 ) . The defects and their temporal pattern of development were similar in all three cases. The earliest manifestations were noticed at age 20 to 40 years and consisted of nasal, slurred speech and progressive ataxia in all four limbs. Severe deterioration occurred over a decade, after which they usually were completely incapacitated and in need of institutional care. General medical examination was uniformly unremarkable except for ortostatic hypotension (triggering of physiological tachycardia). The electrocardiograms were essentially normal. One patient had a tracing with depressed ST segments, probably secondary to sympathicotonia. Symptoms of autonomic nervous dysfunctions were outstanding, with frequent diarrheas and difficulties in emptying the atonic urinary bladder. On neurological examination the following findings were encountered: the optic discs were pale but visual acuity and color vision were normal; the visual fields were
361
HLA-DETERMINATION IN HEREDITARY ATAXIA
intact; pupillary reaction t o light was sluggish; there was n o nystagmus, but voluntary and persuit eye movements were abnormal with ocular dysmetria; caloric responses were weak; speech was slurred and of nasal character; muscle mass and strength seemed uniformly reduced; no stretch reflexes could be elicited though plantar responses were extensor bilaterally ; the gait was clumsy and widely based; on eye closure there was a marked tendency to fall in any direction; finger-nose and knee-heel testings were pathological with pronounced dysmetria and ataxia; vibration and positional senses were defective while threshold to touch, pain and temperature were normal; pes excavatus was another abnormality common to all three patients. Because of pale optic discs, an electroretinogram was obtained and visual evoked responses examined in one patient (woman born 1946, Fig. 5 ) . The results were entirely normal. Electromyographic studies in the same patient confirmed marked sensory neuropathy, with some axonal loss as well. The cerebrospinal fluid was consistently normal with respect t o cytology, protein concentration and electrophoretic protein pattern. Pneumoencephalograms and vertebral angiograms were normal. The patient was once subjected to extensive work-up because of malabsorption due t o hastened intestinal passage, a manifestation of autonomic neuropathy. However, routine laborations in blood (including BIZ and f o l k acid in serum) were normal except for increased sedimentation rates, probably secondary to urinary tract infections. Liver tests did not deviate from normal. Caeruloplasmin in serum and renal excretion of amino acids were also unremarkable.
the microcytotoxicity technique described by Kissmeyer-Nielsen & Kjerbye ( 1967). Lymphocytes from blood samples were purified by flotation on Ficoll-Isopaque (Boyum 1968). HLA antisera were obtained partly through the Scandiatransplant Organization. Fresh or frozen lymphocyte suspensions were used for mixed lymphocyte culture (MLC). Cultures were performed in microplates containing 5 x lo4 responder cells and 5 x lo4 mitomycintreated stimulator cells in a total volume of 0.2 ml medium (Minimal Essential Medium containing Hepes buffer, 10% inactive human AB serum, and penicillin/ streptomycin, 5000 IU of each per ml). Tritiated thymidine ( 1 uCi; specific activity, 25 Ci/mmol) was added t o all cultures on day 5 and incubation continued for 24 h. Cultures were harvested with distilled water in an automatic cell harvester. The cells were absorbed onto glass fiber filters, which were dried at 60°C and thereafter transferred to scintillation vials containing a totuene-based scintillation fluid. Radioactivity was counted in a liquid scintillation detector. MLC results are expressed as counts per minute/culture f SE. Since SE were generally less than 10% of the mean, they are not indicated in the Tables. All cultures were performed in quadriplicate. In all experiments, autologous controls with mitomycin-treated cells (A -t- Am) as well as mixtures of all cells, mitomycintreated (Am + Bm, Am Cm, etc.) were performed.
+
Results
In the first family (Fig. 3 ) all the affected members shared the HLA haplotype Methods A2B12, which was not present in healthy HLA typing was performed according t o members.
362
MOLLER ET AL. Table 1 Results from M L C test performed with cells of family I1
Responder cells
HLA genotype
Disease
11: 1
I I : l (1914) I1 : 3 (1916 I I : 4 (1913 11: 5 (1920) 11: 7 (1921) 111: 3 (1940)
A2B5lA2B5 A2BSIA2B27 A2B61A1988 A 2 B 5 IA2B 27 A2 B 5IA2 B 27 A2B51A2B7
no yes no no yes yes
11 106 123 135 120 147
In the second family (Fig. 4) all the affected members shared the haplotype A2B5, which also was carried by healthy members of the family. The MLC test showed differences both between the affected and the healthy members of the second generation. If an association should exist between the ataxia disease and the HLA region, it can b e speculated that the deceased, affected father was A2B5 homozygous, but that only one of these haplotypes was connected with the disease, since he had only a healthy brother. His wife’s haplotypes must then have been A2B5 and A2B27. If this interpretation is correct the disease would be transmitted by one of the father’s haplotypes A2B5 to the two affected children in the second generation, whereas the father’s other haplotype A2B5, which is not ataxia-linked, was transmitted to the two healthy children. As the disease in this family always has started between 2 0 and 40 years of age i t is probable that the latter two members will remain healthy. The children in the third generation are all between 2 0 and 40 years of age and one of them (born 1954) hasslight, b u t definite symptoms of the disease. In a further two members (born 1 9 4 0 and 1949) subtle symptoms exist. All these members are children of affected parents in the second generation and share the haplotype A2B5 with their affected parents. One
Stimulator cells (cprnlmin x 11 : 3 I1 : 4 I1 : 5 I1 : 7
62 4 92 82 66 30
186 147 24 122 124 153
104 67 92 2 8 102
103 57 99 7 3 103
11: 3
128 38 101 97 92
6
child (born 1947) of an affected parent neither has symptoms nor carries the relevant haplotype A2B5, whereas two healthy members in the third generation have the haplotype A2B5 and are children of healthy members in the second generation. In order to elucidate further the presence of disease-associated HLA haplotypes in this family, an MLC test was performed. The results are shown in Table 1. First, it is obvious that the t w o female siblings in the second generation (born 1 9 2 0 and 1921) are HLA identical, since their cells are mutually non-stimulatory. Secondly, t h e affected man (born 1916) is phenotypically HLA-A and --B identical to his sisters b u t the MLC results show that he is HLA-D heterozygous, since his mitomycin-treated cells give a relative response of more than 50% to responder cells from his three siblings in the second generation. Thus, one interpretation from these data would be that the diseased father in the first generation has thee haplotypes A2B5Da/A2BSDb. His wife has A2B5Dc/A2B27. We shall treat these data below as if the presence of disease in this family were dependent upon the presence of a dominant HLA-linked gene. The third family (Fig. 5 ) has three affected members, who d o not share any specific HLA haplotype. The affected female in the third generation and her
HLA-DETERMINATION IN HEREDITARY ATAXIA
363
Table 2 Results from M L C test performed with cells from family 111 Responder cells
HLA genotype
Disease
1I:l
Stimulator cells (cpmtrnin x II:2 II:3 I I : 4 11:s II:6 III:2 ~~
11: 1 (1904) 11: 2 (1907) 11: 3 (1909)
II:4 11:s (1916)
II:6 (1918) II1:2 (1946) control
A25 B 1 3lA25 B27 A25B131A2B14 A2 5 B 1 3 /A2 B 1 4 A1 B8lA28B12 A2 5 B 1 3lA2 5 B2 7 A25B13IA25B27 A1 B8IA2B14
3 n.t. 20 44 16
5 24 15
mother share the haplotype A2B14, but the other affected woman in the second generation lacks this haplotype. In addition, two more healthy members of the second generation have A2B14. All siblings in the second generation share the parental haplotype Aw25B13. MLC tests were performed in this family t o investigate whether the A25B13 haplotype was identical in all sibIings and to study the possible existence of HLA-B-D recombinations. The data are shown in Table 2. Thus, the individuals born 1907, 1 9 0 9 and 1916 were HLA identical. Another HLA identical couple was sibs born 1904 and 1918. This supports the idea that all the siblings have inherited an identical A25B13 haplotype from one of the parents. Unfortunately, we have been unable to test any other members of this large family to try and establish the specific haplotypes of the diseased father of the proband in the second generation.
Discussion The disease in different families with hereditary ataxia has its own features. In the first family, symptoms included a sensory polyneuropathy with bladder disturbances and peculiar pains, in the second family mental retardation and in the third
n.t. 10 5 31 7 25 29 18
32 8
3 21 5 27 27 17
86 17 25 2 38 24 24 20
35 7 3 30 3 3 16 16
2 11 19 21 20 4 36 20
control ~
40 20 18 24 23 30 2 22
5 12 18 16 13 28 27 3
family vegetative symptoms. Therefore, it is possible that the disease is caused by genes in different loci in different families. Jackson e t al. (1977) recently reported on HLA studies in a large family with SCA in which 1 7 near relatives of the proband were investigated; 1 6 of these had the disease. The haplotype A3B14 was present in four of the proband’s children, all affected by the disease, whereas the other haplotype A2B15 was present in one healthy child and in one child with disease. If the disease predisposing gene is HLA linked, the A2B15 child with disease must have a recombinant chromosome. The total Lod score for HLA linkage in the study was 3.15, which strongly argues in favour of the theory that the autosomal dominant gene responsible for development of disease in their family was present o n the 6th chromosome, linked to, bur nor present within, the HLA region. Let us accept their data as correct and study in detail our own results in the light of theirs. Thus, in the first family, one of the haplotypes of the proband in the second generation must carry the SCA disease gene. The results agree well with this postulate, the two affected daughters who have SCA inherited one of the mother’s haplotypes, A2B12, whereas their healthy sister inherited the A2B15 haplotype from
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the proband. The results in this family are similar to those reported by Yakura e t al. (1974), where it was found that three SCA sibs shared one HLA haplotype, A9B5. In the second family, there is a preponderance of the HLA haplotype A2B5 in the SCA patients. However, there are probably three distinct A2B5 haplotypes in this family, of which only one is tentatively associated with the gene for SCA. Thus, we shall argue that this haplotype is present in those members of the second generation who have the disease, but absent from those not affected. On the other hand, this is not borne out from the data, since the two affected siblings are MLC incompatible, whereas the affected sister is MLC compatible with her healthy sister. Consequently, we must assume that a recombinational event has occurred in the paternal haplotype of the patient born 1921 so that she inherited the SCA gene with her A2B5 haplotype. This is consistent with the findings that her two sons who inherited this haplotype most probably have the disease. Furthermore, of her affected brother’s two children, one son inherited the haplotype and also probably has the disease whereas the daughter, who inherited the other haplotype, which is presumably not disease associated, is healthy. Finally, the healthy brother in the second generation (born 1914) inherited one A2B5 haplotype from his healthy mother, and there is a 50% chance that his paternal haplotype A2B5 is not associated with the SCA gene. Thus, his two children would be, and are, healthy. The data from the last family are not directly compatible with close HLA linkage of the SCA gene. The inheritance pattern is clearly autosomal and dominant when the complete family pedigree is analyzed. As mentioned above, all first generation sibs
inherited the A25B13 haplotype, presumably from the mother. Should disease in this family be associated with a particular HLA haplotype, it would probably not be one shared by all sibs, since four are healthy and only two have the disease. Therefore, we must assume that one of the supposedly paternal haplotypes, A2B14 or A25B27, is linked to the SCA gene. The affected woman and her daughter, who have the disease, share the A2B14 haplotype. Let us therefore assume that the disease gene is present on this A2B14 chromosome. But then, a further two siblings (born 1907 and 1909) who have this chromosome are healthy. The other haplotype A25B27 is present in the other affected sibling (born 1918). However, it is conceivable that the proband (born 1916) could have received a paternal recombinant sixth chromosome which led to propagation of the disease in her daughter. If this were true, one also has to assume that the first generation sib (born 1904) received a recombinant chromosome A2SB27, since she is healthy. The findings of Jackson et al. (1977) are consistent with the position of the SCA gene a t a distance of approximately 12 cM from the A o r B side of the HLA region. We are, in this family, assuming a recombination frequency of 216 from the A or D side of HLA, which is not quite inconceivable in the light of Jackson’s data. However, we would like to point out that in families I and 11, HLA linkage for the SCA gene is more likely. Since close linkage to the HLA region is not present in patients with SCA, it is likely that associations between a particular HLA allele and SCA will not be found in randomly chosen non-related patients. This is important t o point out, since only family studies will be able to tell in those cases
HLA-DETERMINATION IN HEREDITARY ATAXIA
whether a gene which is associated with the jevelopment of a particular disease will be present on the sixth chromosome or not. Consequently, the gene which is responsible for the development of SCA is most probably an allele of a locus distinct from the HLA linked loci which contain genes that predispose individuals to the development of other neurological disorders such as multiple sclerosis (Alter et al. 1976, Olsson e t al. 1976) and myasthenia gravis (Fog et al. 1977). Our data presented above, and our interpretations of these data can be considered to be consistent with, but do not prove, the existence of an SCA gene on the sixth chromosome. We have extended the data of Jackson et al. (1977) and shown that the SCA gene is not present within the B-D part of the HLA region. This was, however, not t o be expected since their data indicated a distance of 12 cM from the HLA region to the SCA locus. For a future analysis of the position of the SCA gene, determinations of PGMj and PGs alleles in the family might prove informative, since the PG5 locus is thought t o be present.., 20 cM on the distant side of the HLA region and PGM3 -15 cM to the centromere part of the sixth chromosome, (Lamm e t al. 1972, Jongsma et al. 1973, van Someren et al. 1974 (for reference see Bach & van Rood 1966)). References Alter, M., Harshe, M. & Andersson E. V. (1976) Genetic association of multiple sclerosis and HL-A determinants. Neurology 26, 3136. Bach, F. H. & van Rood, J. J. (1976) The major histocompatibility complex-genetics and biology. New Engl. J. Med. 295, 927-936. Behan, P. O., Simpson, J. A. & Dicke, A. (1973) Immune response genes in myasthenia gravis. Lancet ii, 1033.
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B e r t r a m , I. & Kuwert, E. (1972) HL-A antigen frequencies in multiple sclerosis. Eur. J . Neurol. 7 , 74-78. Boyum, A. (1968) Separation of leukocytes from blood and bone marrow. Scand. J. Clin. Lab. Invest. 21 (suppl. 97). 1-189. Fog, T., Schuller, E. & Jersild, C. (1977) Myasthenia gravis. In HLA and Disease, eds. Dausset, J. & Svejgaard, A, pp.119-122. Munksgaard: Copenhagen. Jackson, J., Currier, R., Terasaki, P. & Morton, N. (1977) Spinocerebellar ataxia and HLA linkage. New Engl. J , Med. 296, 11 38-1 141. Jersild, C., Svejgaard, A. & Fog, T. (1972) HL-A antigens and multiple sclerosis. Lancet 1, 1240-1241. Jongsma, A., van Someren, H., Westerveld, A., Hagenmeier, A. & Pearson, P. (1973) Localization of genes o n human chromosomes using human Chinese hamster somatic cell hybrids. Assignment of PGM, t o chromosome C6 and regional mapping of the PGD, PGM, and Pep-C genes on chromosome A l . Humangenetik 20, 195-202. Kissmeyer-Nielsen, F. & Kjerbye, K. E. (1967) Lymphocytotoxic microtechnique: Purification of lymphocytes by flotation. In Histocompatibility Testing (1967). ed. Curtoni, E. S. pp. 381-383. Munksgaard: Copenhagen. Lamm, L. U., Kissmeyer-Nielsen, F., Svejgaard, A., Bruun-Petersen, G., Thorsby, E., Mayr, W. & Hogman, C. (1972) On the orientation of the HLA-A region and the PGM, locus in the chromosome. Tissue Antigens. 2, 205-214. Naito, S., Namerow, N. & Mickey, M. (1972) Multiple sclerosis: Association with HL-A. Tissue Antigens 2, 1-4. Olsson, J. E., Moller, E. & Link, H. (1976) HLA haplotypes in families with high frequency of multiple sclerosis. Arch Neurol. 33, 808-812. Pirskanen, R., Tiilikainen, A. & Hokkanen, E. (1972) Histocompatibility (HLA-A) antigens associated with myasthenia gravis. Ann Clin. Hes. 4, 304-306. van Someren, H., Westerveld, A., Hagemeier, A., Mees, J. R., Meera Khan, P. & Saalberg, 0. B. (1974) Human antigen and enzyme markers in man-Chinese hamster somatic cell hybrids. Evidence for synteny between the HL-A, PGM,, Me, and IPO-B loci. Proc. Nut. Acad. Sci. USA 71,962-965.
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Yakura, H., Wakisaka, A. & Fumimoto, S . (1974) Address: Hereditary ataxia and HL-A genotypes. New. Jan-Edvin Olsson, M . D Dept. Neurology Engl. J . M e d . 291, 154-155. University Hospital S-22185Lund Sweden
HLA-Determination in Families with Hereditary Ataxia Erna Moller, Bengt Hindfelt and * Jan-Edvin Olsson ' Department of Clinical Immunology, Karolinska Institute, Huddinge Hospital, Huddinge and ' Department of Neurology, University Hospital, Lund, Sweden
In three families with hereditary ataxia, where the inheritance pattern was autosomal and dominant, HLA antigens were determined in 25 members. In two of the families, HLA linkage of disease was suggested, whereas in the third family, the data did not directly support this concept, since two recornbinational events between the postulated locus for disease and t h e HLA region had t o be assumed. However, with this assumption, our data are compatible with those of one family described recently (Jackson e t al. 1977) implying the presence o n the sixth chromosome, outside the HLA region, of a locus that determines the development of spino cerebellar ataxia (SCA). Further tests with definition of enzyme markers will have to be performed before conclusions as t o HLA linkage of a postulated SCA gene can be made. Received for publication 6 April, revised, accepted 2 June 1978
Several neurological disorders with uncertain mode of inheritance are associated with genetic markers of the HLA system, This is the case for multiple sclerosis (MS) (Bertrams & Kuvert 1972, Jersild e t al. 1972, Naito e t al. 1972) and myasthenia gravis (MG) (Pirskanen et al. 1972, Behan et al. 1973). Certain neurological diseases, e.g. the hereditary ataxias, have a pattern of inheritance which can be classified according t o clinical observations. However, even with a dominant inheritance, it is impossible t o predict which members of the family will subsequently develop clinical symptoms. In most cases this happens when the patients have grown up and already have children themselves. Since there is no treatment for these disorders it is essential to give genetic information and thereby prevent transmittance of the
disease. According t o Jackson et al. (1977) an ataxia locus is connected with the HLA locus on the sixth chromosome. The aim of our study was to prove whether or not determination of the HLA antigens is of clinical-genetic value in families with hereditary ataxias, where the circumstances indicate a dominant inheritance. Material Patients HLA antigens were determined in 25 members of three families with spinocerebellar ataxia(SCA) where the inheritance pattern was autosomal and dominant. In the first family (Fig. 1) several members were affected with a disorder consisting of a peculiar lower back and abdominal pain, defects of micturation and severe ataxia. Three affected members were examined.
MOLLER ET AL.
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The onset of the disorder was usually between the ages of 20 and 3 5 . The initial symptom was a severe low back pain, radiating over the lower abdomen and down the inquinal regions. Concurrently, urinary urgency was conspicious. The symptoms were initially interpreted as being due t o urinary infections and treated accordingly without success. Subsequently the pain became sustained and incapacitating. Difficulties in emptying the bladder gradually evolved, necessitating the continuous use of an indwelling catheter. Within a few years, a progressive limbataxia appeared, predominantly in the legs. The affected members have all been of a slender body constitution. Somatic examination has been unremarkable, with the exceptions mentioned above. On neurological examination limb ataxia and a wide based gait disturbance were the dominating features. Cranial nerves were intact, apart from a slurred speech and some horizontal nystagmus. Intermittently, head tremor was observed. There were no pareses, although muscle mass seemed reduced. Stretch reflexes could not be elicited and plantar responses were flexor. Appreciation of touch, pain and temperature was normal, while vibration and positional senses were severely defective. As in other reported families, extensive laboratory tests on blood and cerebrospinal
0
A
Figure 1. Pedigrees of two families with autosomal dominant spinocerebellar ataxia. Left family I, right family 11.
fluid were non-contributory in elucidating the underlying pathogenesis. Myelographic studies did not reveal any morphological explanation t o the intense pain experienced early in the course. Despite normal neuroophthalmological findings, visual evoked responses revealed delayed lathencies (160-180 ms). Electromyography was consistent with a predominantly sensory neuropathy, leaving very few myelinated fibers intact. During the period of investigation, one of the affected siblings--born 1915-in the second generation died. At autopsy classical pathological features were found with demyelination of the dorsal spinal columns and the dorsal spinal ganglions. Degenerative changes were observed within the dentate nucleus, the Purkinje cells and the frontal cerebral cortex. The lumbo-sacral nerve roots were considerably smaller than normal, surrounded by fibrous tissue and exhibiting inflammatory infiltration. The full neurological syndrome affecting members of the second family (Fig. 1) was almost identical to that of the third family (Fig. 2), but the families were unrelated as far as could be traced. The earliest manifestations were observed at about the same age (20 to 40 years) and consisted of slurred speech and a progressive, ataxic gait disturbance. The temporal profile of neurological deterioration was also similar
HLA-DETERMINATION IN HEREDITARY ATAXIA
..
I
359
rrrLlA
0
0 0 0 0 0 .
00 NORMAL
MALE FEMALE AFFECTED MALE. FEMALE DECEASED AS CHILDREN
4
Figure 2. Pedigree of one family (111) with autosomal dominant spinocerebellar ataxia.
I
111
6-40
4-45
6-41
A2 1 81218
A2 9 812140
09 NORMAL W 0
0
MALE FEMALE AFFECTED MALE, FEMALE DECEASED
Figure 3. Pedigree showing the HLA haplotypes in family 1. HLA-A and -B antigens are shown in haplotype form separated with vertical lines.
7-
I
5
0 NORMAL 0
MALE. FEMALE AFFECTED MALE, FEMALE DECEASED
Figure 4. Pedigree showing the tentative HLA haplotypes in family 11. HLA-A, - B and are indicated as in Fig. 3 .
-D
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MOLLER ET AL.
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?
A2
AFFECTED MALE. FEMALE DECEASED DECEASED AS CHILDREN NOT INVESTIGATED
Figure 5. Pedigree showing the HLA haplotypes in family 111.
in the two families. Medical examination was unrevealing, except for a tendency in some members of the family to develop moderate arterial hypertension. The neurological findings were identical in all respects to those encountered in the third family. Two examined members of the family (man born 1916 and woman born 1921, Fig. 4) presented the complete syndrome, while one male - borne 1954 -in the third generation (Fig. 4)exhibited ataxia on finger-nose and knee-heel testing. Another two male relatives in the third generation - born 1940 and 1949 (Fig. 4)-had somewhat equivocal findings, i.e. minor ataxia or tremor, weakened or absent Achilles reflexes. These subtle findings may indicate that they too are affected and perhaps will develop more clear-cut defects with time. Extensive laboratory tests on blood and cerebrospinal fluid in the affected members have not provided any clue to the underlying metabolic abnormality. Pneumoencephalogram in the man born 1916 has revealed a widened major cistern and an enlarged fourth ventricle, indicating an atrophic process. In the third family (Fig.
2) a neurological syndrome with features of Friedreich ataxia and sensory polyneuropathy has affected three females, presently alive and examined (Fig. 5 ) . The defects and their temporal pattern of development were similar in all three cases. The earliest manifestations were noticed at age 20 to 40 years and consisted of nasal, slurred speech and progressive ataxia in all four limbs. Severe deterioration occurred over a decade, after which they usually were completely incapacitated and in need of institutional care. General medical examination was uniformly unremarkable except for ortostatic hypotension (triggering of physiological tachycardia). The electrocardiograms were essentially normal. One patient had a tracing with depressed ST segments, probably secondary to sympathicotonia. Symptoms of autonomic nervous dysfunctions were outstanding, with frequent diarrheas and difficulties in emptying the atonic urinary bladder. On neurological examination the following findings were encountered: the optic discs were pale but visual acuity and color vision were normal; the visual fields were
361
HLA-DETERMINATION IN HEREDITARY ATAXIA
intact; pupillary reaction t o light was sluggish; there was n o nystagmus, but voluntary and persuit eye movements were abnormal with ocular dysmetria; caloric responses were weak; speech was slurred and of nasal character; muscle mass and strength seemed uniformly reduced; no stretch reflexes could be elicited though plantar responses were extensor bilaterally ; the gait was clumsy and widely based; on eye closure there was a marked tendency to fall in any direction; finger-nose and knee-heel testings were pathological with pronounced dysmetria and ataxia; vibration and positional senses were defective while threshold to touch, pain and temperature were normal; pes excavatus was another abnormality common to all three patients. Because of pale optic discs, an electroretinogram was obtained and visual evoked responses examined in one patient (woman born 1946, Fig. 5 ) . The results were entirely normal. Electromyographic studies in the same patient confirmed marked sensory neuropathy, with some axonal loss as well. The cerebrospinal fluid was consistently normal with respect t o cytology, protein concentration and electrophoretic protein pattern. Pneumoencephalograms and vertebral angiograms were normal. The patient was once subjected to extensive work-up because of malabsorption due t o hastened intestinal passage, a manifestation of autonomic neuropathy. However, routine laborations in blood (including BIZ and f o l k acid in serum) were normal except for increased sedimentation rates, probably secondary to urinary tract infections. Liver tests did not deviate from normal. Caeruloplasmin in serum and renal excretion of amino acids were also unremarkable.
the microcytotoxicity technique described by Kissmeyer-Nielsen & Kjerbye ( 1967). Lymphocytes from blood samples were purified by flotation on Ficoll-Isopaque (Boyum 1968). HLA antisera were obtained partly through the Scandiatransplant Organization. Fresh or frozen lymphocyte suspensions were used for mixed lymphocyte culture (MLC). Cultures were performed in microplates containing 5 x lo4 responder cells and 5 x lo4 mitomycintreated stimulator cells in a total volume of 0.2 ml medium (Minimal Essential Medium containing Hepes buffer, 10% inactive human AB serum, and penicillin/ streptomycin, 5000 IU of each per ml). Tritiated thymidine ( 1 uCi; specific activity, 25 Ci/mmol) was added t o all cultures on day 5 and incubation continued for 24 h. Cultures were harvested with distilled water in an automatic cell harvester. The cells were absorbed onto glass fiber filters, which were dried at 60°C and thereafter transferred to scintillation vials containing a totuene-based scintillation fluid. Radioactivity was counted in a liquid scintillation detector. MLC results are expressed as counts per minute/culture f SE. Since SE were generally less than 10% of the mean, they are not indicated in the Tables. All cultures were performed in quadriplicate. In all experiments, autologous controls with mitomycin-treated cells (A -t- Am) as well as mixtures of all cells, mitomycintreated (Am + Bm, Am Cm, etc.) were performed.
+
Results
In the first family (Fig. 3 ) all the affected members shared the HLA haplotype Methods A2B12, which was not present in healthy HLA typing was performed according t o members.
362
MOLLER ET AL. Table 1 Results from M L C test performed with cells of family I1
Responder cells
HLA genotype
Disease
11: 1
I I : l (1914) I1 : 3 (1916 I I : 4 (1913 11: 5 (1920) 11: 7 (1921) 111: 3 (1940)
A2B5lA2B5 A2BSIA2B27 A2B61A1988 A 2 B 5 IA2B 27 A2 B 5IA2 B 27 A2B51A2B7
no yes no no yes yes
11 106 123 135 120 147
In the second family (Fig. 4) all the affected members shared the haplotype A2B5, which also was carried by healthy members of the family. The MLC test showed differences both between the affected and the healthy members of the second generation. If an association should exist between the ataxia disease and the HLA region, it can b e speculated that the deceased, affected father was A2B5 homozygous, but that only one of these haplotypes was connected with the disease, since he had only a healthy brother. His wife’s haplotypes must then have been A2B5 and A2B27. If this interpretation is correct the disease would be transmitted by one of the father’s haplotypes A2B5 to the two affected children in the second generation, whereas the father’s other haplotype A2B5, which is not ataxia-linked, was transmitted to the two healthy children. As the disease in this family always has started between 2 0 and 40 years of age i t is probable that the latter two members will remain healthy. The children in the third generation are all between 2 0 and 40 years of age and one of them (born 1954) hasslight, b u t definite symptoms of the disease. In a further two members (born 1 9 4 0 and 1949) subtle symptoms exist. All these members are children of affected parents in the second generation and share the haplotype A2B5 with their affected parents. One
Stimulator cells (cprnlmin x 11 : 3 I1 : 4 I1 : 5 I1 : 7
62 4 92 82 66 30
186 147 24 122 124 153
104 67 92 2 8 102
103 57 99 7 3 103
11: 3
128 38 101 97 92
6
child (born 1947) of an affected parent neither has symptoms nor carries the relevant haplotype A2B5, whereas two healthy members in the third generation have the haplotype A2B5 and are children of healthy members in the second generation. In order to elucidate further the presence of disease-associated HLA haplotypes in this family, an MLC test was performed. The results are shown in Table 1. First, it is obvious that the t w o female siblings in the second generation (born 1 9 2 0 and 1921) are HLA identical, since their cells are mutually non-stimulatory. Secondly, t h e affected man (born 1916) is phenotypically HLA-A and --B identical to his sisters b u t the MLC results show that he is HLA-D heterozygous, since his mitomycin-treated cells give a relative response of more than 50% to responder cells from his three siblings in the second generation. Thus, one interpretation from these data would be that the diseased father in the first generation has thee haplotypes A2B5Da/A2BSDb. His wife has A2B5Dc/A2B27. We shall treat these data below as if the presence of disease in this family were dependent upon the presence of a dominant HLA-linked gene. The third family (Fig. 5 ) has three affected members, who d o not share any specific HLA haplotype. The affected female in the third generation and her
HLA-DETERMINATION IN HEREDITARY ATAXIA
363
Table 2 Results from M L C test performed with cells from family 111 Responder cells
HLA genotype
Disease
1I:l
Stimulator cells (cpmtrnin x II:2 II:3 I I : 4 11:s II:6 III:2 ~~
11: 1 (1904) 11: 2 (1907) 11: 3 (1909)
II:4 11:s (1916)
II:6 (1918) II1:2 (1946) control
A25 B 1 3lA25 B27 A25B131A2B14 A2 5 B 1 3 /A2 B 1 4 A1 B8lA28B12 A2 5 B 1 3lA2 5 B2 7 A25B13IA25B27 A1 B8IA2B14
3 n.t. 20 44 16
5 24 15
mother share the haplotype A2B14, but the other affected woman in the second generation lacks this haplotype. In addition, two more healthy members of the second generation have A2B14. All siblings in the second generation share the parental haplotype Aw25B13. MLC tests were performed in this family t o investigate whether the A25B13 haplotype was identical in all sibIings and to study the possible existence of HLA-B-D recombinations. The data are shown in Table 2. Thus, the individuals born 1907, 1 9 0 9 and 1916 were HLA identical. Another HLA identical couple was sibs born 1904 and 1918. This supports the idea that all the siblings have inherited an identical A25B13 haplotype from one of the parents. Unfortunately, we have been unable to test any other members of this large family to try and establish the specific haplotypes of the diseased father of the proband in the second generation.
Discussion The disease in different families with hereditary ataxia has its own features. In the first family, symptoms included a sensory polyneuropathy with bladder disturbances and peculiar pains, in the second family mental retardation and in the third
n.t. 10 5 31 7 25 29 18
32 8
3 21 5 27 27 17
86 17 25 2 38 24 24 20
35 7 3 30 3 3 16 16
2 11 19 21 20 4 36 20
control ~
40 20 18 24 23 30 2 22
5 12 18 16 13 28 27 3
family vegetative symptoms. Therefore, it is possible that the disease is caused by genes in different loci in different families. Jackson e t al. (1977) recently reported on HLA studies in a large family with SCA in which 1 7 near relatives of the proband were investigated; 1 6 of these had the disease. The haplotype A3B14 was present in four of the proband’s children, all affected by the disease, whereas the other haplotype A2B15 was present in one healthy child and in one child with disease. If the disease predisposing gene is HLA linked, the A2B15 child with disease must have a recombinant chromosome. The total Lod score for HLA linkage in the study was 3.15, which strongly argues in favour of the theory that the autosomal dominant gene responsible for development of disease in their family was present o n the 6th chromosome, linked to, bur nor present within, the HLA region. Let us accept their data as correct and study in detail our own results in the light of theirs. Thus, in the first family, one of the haplotypes of the proband in the second generation must carry the SCA disease gene. The results agree well with this postulate, the two affected daughters who have SCA inherited one of the mother’s haplotypes, A2B12, whereas their healthy sister inherited the A2B15 haplotype from
364
MOLLER ET AL.
the proband. The results in this family are similar to those reported by Yakura e t al. (1974), where it was found that three SCA sibs shared one HLA haplotype, A9B5. In the second family, there is a preponderance of the HLA haplotype A2B5 in the SCA patients. However, there are probably three distinct A2B5 haplotypes in this family, of which only one is tentatively associated with the gene for SCA. Thus, we shall argue that this haplotype is present in those members of the second generation who have the disease, but absent from those not affected. On the other hand, this is not borne out from the data, since the two affected siblings are MLC incompatible, whereas the affected sister is MLC compatible with her healthy sister. Consequently, we must assume that a recombinational event has occurred in the paternal haplotype of the patient born 1921 so that she inherited the SCA gene with her A2B5 haplotype. This is consistent with the findings that her two sons who inherited this haplotype most probably have the disease. Furthermore, of her affected brother’s two children, one son inherited the haplotype and also probably has the disease whereas the daughter, who inherited the other haplotype, which is presumably not disease associated, is healthy. Finally, the healthy brother in the second generation (born 1914) inherited one A2B5 haplotype from his healthy mother, and there is a 50% chance that his paternal haplotype A2B5 is not associated with the SCA gene. Thus, his two children would be, and are, healthy. The data from the last family are not directly compatible with close HLA linkage of the SCA gene. The inheritance pattern is clearly autosomal and dominant when the complete family pedigree is analyzed. As mentioned above, all first generation sibs
inherited the A25B13 haplotype, presumably from the mother. Should disease in this family be associated with a particular HLA haplotype, it would probably not be one shared by all sibs, since four are healthy and only two have the disease. Therefore, we must assume that one of the supposedly paternal haplotypes, A2B14 or A25B27, is linked to the SCA gene. The affected woman and her daughter, who have the disease, share the A2B14 haplotype. Let us therefore assume that the disease gene is present on this A2B14 chromosome. But then, a further two siblings (born 1907 and 1909) who have this chromosome are healthy. The other haplotype A25B27 is present in the other affected sibling (born 1918). However, it is conceivable that the proband (born 1916) could have received a paternal recombinant sixth chromosome which led to propagation of the disease in her daughter. If this were true, one also has to assume that the first generation sib (born 1904) received a recombinant chromosome A2SB27, since she is healthy. The findings of Jackson et al. (1977) are consistent with the position of the SCA gene a t a distance of approximately 12 cM from the A o r B side of the HLA region. We are, in this family, assuming a recombination frequency of 216 from the A or D side of HLA, which is not quite inconceivable in the light of Jackson’s data. However, we would like to point out that in families I and 11, HLA linkage for the SCA gene is more likely. Since close linkage to the HLA region is not present in patients with SCA, it is likely that associations between a particular HLA allele and SCA will not be found in randomly chosen non-related patients. This is important t o point out, since only family studies will be able to tell in those cases
HLA-DETERMINATION IN HEREDITARY ATAXIA
whether a gene which is associated with the jevelopment of a particular disease will be present on the sixth chromosome or not. Consequently, the gene which is responsible for the development of SCA is most probably an allele of a locus distinct from the HLA linked loci which contain genes that predispose individuals to the development of other neurological disorders such as multiple sclerosis (Alter et al. 1976, Olsson e t al. 1976) and myasthenia gravis (Fog et al. 1977). Our data presented above, and our interpretations of these data can be considered to be consistent with, but do not prove, the existence of an SCA gene on the sixth chromosome. We have extended the data of Jackson et al. (1977) and shown that the SCA gene is not present within the B-D part of the HLA region. This was, however, not t o be expected since their data indicated a distance of 12 cM from the HLA region to the SCA locus. For a future analysis of the position of the SCA gene, determinations of PGMj and PGs alleles in the family might prove informative, since the PG5 locus is thought t o be present.., 20 cM on the distant side of the HLA region and PGM3 -15 cM to the centromere part of the sixth chromosome, (Lamm e t al. 1972, Jongsma et al. 1973, van Someren et al. 1974 (for reference see Bach & van Rood 1966)). References Alter, M., Harshe, M. & Andersson E. V. (1976) Genetic association of multiple sclerosis and HL-A determinants. Neurology 26, 3136. Bach, F. H. & van Rood, J. J. (1976) The major histocompatibility complex-genetics and biology. New Engl. J. Med. 295, 927-936. Behan, P. O., Simpson, J. A. & Dicke, A. (1973) Immune response genes in myasthenia gravis. Lancet ii, 1033.
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B e r t r a m , I. & Kuwert, E. (1972) HL-A antigen frequencies in multiple sclerosis. Eur. J . Neurol. 7 , 74-78. Boyum, A. (1968) Separation of leukocytes from blood and bone marrow. Scand. J. Clin. Lab. Invest. 21 (suppl. 97). 1-189. Fog, T., Schuller, E. & Jersild, C. (1977) Myasthenia gravis. In HLA and Disease, eds. Dausset, J. & Svejgaard, A, pp.119-122. Munksgaard: Copenhagen. Jackson, J., Currier, R., Terasaki, P. & Morton, N. (1977) Spinocerebellar ataxia and HLA linkage. New Engl. J , Med. 296, 11 38-1 141. Jersild, C., Svejgaard, A. & Fog, T. (1972) HL-A antigens and multiple sclerosis. Lancet 1, 1240-1241. Jongsma, A., van Someren, H., Westerveld, A., Hagenmeier, A. & Pearson, P. (1973) Localization of genes o n human chromosomes using human Chinese hamster somatic cell hybrids. Assignment of PGM, t o chromosome C6 and regional mapping of the PGD, PGM, and Pep-C genes on chromosome A l . Humangenetik 20, 195-202. Kissmeyer-Nielsen, F. & Kjerbye, K. E. (1967) Lymphocytotoxic microtechnique: Purification of lymphocytes by flotation. In Histocompatibility Testing (1967). ed. Curtoni, E. S. pp. 381-383. Munksgaard: Copenhagen. Lamm, L. U., Kissmeyer-Nielsen, F., Svejgaard, A., Bruun-Petersen, G., Thorsby, E., Mayr, W. & Hogman, C. (1972) On the orientation of the HLA-A region and the PGM, locus in the chromosome. Tissue Antigens. 2, 205-214. Naito, S., Namerow, N. & Mickey, M. (1972) Multiple sclerosis: Association with HL-A. Tissue Antigens 2, 1-4. Olsson, J. E., Moller, E. & Link, H. (1976) HLA haplotypes in families with high frequency of multiple sclerosis. Arch Neurol. 33, 808-812. Pirskanen, R., Tiilikainen, A. & Hokkanen, E. (1972) Histocompatibility (HLA-A) antigens associated with myasthenia gravis. Ann Clin. Hes. 4, 304-306. van Someren, H., Westerveld, A., Hagemeier, A., Mees, J. R., Meera Khan, P. & Saalberg, 0. B. (1974) Human antigen and enzyme markers in man-Chinese hamster somatic cell hybrids. Evidence for synteny between the HL-A, PGM,, Me, and IPO-B loci. Proc. Nut. Acad. Sci. USA 71,962-965.
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Yakura, H., Wakisaka, A. & Fumimoto, S . (1974) Address: Hereditary ataxia and HL-A genotypes. New. Jan-Edvin Olsson, M . D Dept. Neurology Engl. J . M e d . 291, 154-155. University Hospital S-22185Lund Sweden
