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Structural integrity of the human albumin gene in congenital analbuminemia
Vol. 116, No. 3, 1983
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pages 817-821
November 15, 1983
STRUCIUPAL INTF~PIq~f OF ~ H79iAN ALBUMIN G ~ IN CONGEb~TAL 7~A]NB[~IIK~IA
Robert A. Avery.1 , Elliot Alpert 2 , Kurt ~ i g a n d 3 , and Achilles Dugaiczyk I'4 I)
Department of Cell Biology, Bavlor College of Medicine, Houston, Texas 77030 2) C~stroenterologyDivision, Department of Medicine, Baylor College of Medicine, Houstmn, T~xas 77030 3) Department of Clinical Pharmacology, [~iversity of Berne, Berne,~tzerland 4) Department of Biochemist~,, University of California, Riverside, California 92521 Received September 9, 1983 The human serum albumin gene was analyzed by restriction endonuclease mapping of chromosomal DNA isolated from a patient with congenital analbuminemia. Following digestion with a variety of restriction e_ndonucleases, tJne DNA from this individual produced the same fragments with homology to a serum albumin cDNA probe as did a control DNA specimen. Therefore, the genetic condition of congenital ana]buminemia is not caused by any gross structural rearrange]nent or deletion of the gene itself, but may result from an abnormality in the gene's fine structure, perhaps affece~ing regulation or processing of the primary RNA transcript.
Alb%mtin normally accounts for about 60 percent of the total serum proteins and helps to maintain osmotic pressure, and to transport ions, fatty acids, metabolites, hormones, and drugs (1). However, in the rare genetically transmitted disorder of analbt~ninemia, only trace amounts of this protein are detectable in the serum.
Despite albumin's varied functions, its
virtua]Iv complete absence produces surprisingly few ~%ynlotoms. Edema is present in most of the 22 reported cases, but other serious con~lications are not present.
The increased level of other plasma proteins which usually
acco~oanies analbuminemia partially compensates for the lack of albumin, alleviating the consequences of its absence (2). In an effort to characterize the molecular nature of this genetic abnormality, we have compared the gross structure of the serum albumin gene Send correspondence to: Dr. Elliot Alpert, Gastroenterology Section, Department of Medicine, 6565 Fannin, B-503 , Houston, Texas 77030
0006-291 X/83 $1.50 817
Copyright © 1983 by Academic Press, Inc. All rights o f reproduction in any form reserved.
Vol. 116, No. 3, 1 9 8 3
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
from an analbuminemic and control individual by restriction endonuclease analysis.
This study has been made possible by isolation of DNA from blood
of a newly discovered case of analbumine/nia, and bs~ the availability of two human serum albuntin cDNA clones which were constructed and sequenced in this laboratory (3). These two overlapping clones, pHA36 and pH/k2.06, encode 2.1 kb of hums~m serum albumin mRkih seq~lence fro~ the 3' poly-(A) tail to within 62 bases of the CAP site.
The entire amino acid sequence of albumin is
encoded on this 2.1 P~ stretch of cD~iA, which in turn is d i s ~ r s e d into multiple exons over a region of 16 kb of chromosomal D}IA (4) located on qli-22 of human chromosome 4 (5).
MATERTALS AND ~ T H O D S Blood was obtained from a 60 year old Swiss nmn (6). }Iigh molecular weight D~IA was isolated from peripheral blood ]eukocytes by tJne method of Goosens and Kan (7) , except that the red blood cells were eliminated ~! lysis with ammonium chloride (8) befre the white blood cells were digested with proteinase K. TweDty ~g aliquots of this analbuminemic DNA and of normal htmmn DNA were digested to conpletion with AvaII, HindIII, HpaI, MspI, EcoRI, and SstI restriction endonucleases according to the suppliers' (New England Biolabs and Bethesda Research Ix~boratories) instructions. The resulting fragments were separated by electrophoresis through a 1% agarose gel (9) and transferred to a nitrocellulose paper (Schleicher & Schuell, BAd5) by Sou%/nern' s procedure (I0). The probes, ~ 6 stud pHA206, were nick-translated (Ii) to a sDecific activity of I0- [~ p] cpm/~g DNA and then hybridized to the DNA-containing nitrocellulose paper in a solution containing 0.2% SDS, 5xSSPE (SSPE = 0.18 M NaCl, 10ram sodium phosphate, 1ram ~IYPA; pH 7.0) and 5x Demhardt's solution (12) for 18 hrs at 68°C. After the filter was washed at 68°C with 2xSSPE and dried, it was autoradiographed wit/n Dupond Cronex film and an intensifying screen. RESULTS Fig. 1 shows the electrophoretic separation of serum proteins from a control and the analbuminemic individual analyzed in the present study. There is a striking absence of any protein in the region of the most prominent albumin band from the control serum.
However, it may be
interesting to note that a trace amount of albumin (1.7 rag/100 ml; normal concentration:
4.2
g/100 ml) was found (6) in our analbuminemic patient by
specific inrmlnoprecipitation,
but it had a slightly different migration
velocity than normal albumin when subjected to polyacrvlamide gel
818
Vol. 116, No. 3, 1 9 8 3
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS 1 2 3 4 5 6 N A - N A - N A - N A - N A-N A-
i
2
Kb
®
® Fig. 1
Cellulose acetate electrophoretic separation of serum proteins from a control (lane I) and the analbuminemic serum (lane 2) used in the present study.
Fig. 2
Autoradiogram of_restriction endonucl~@se digested normal (N) and analbtmd_nemic (A) DNA hybridized to --P-labelled human albtmlin cDNA clones pHA36 and pHA206 (3). The DNA in the two lanes under heading i have been digested with AvaII. Similarly, headings 2,3,4,5, and 6, correspond to diges-~{ons with HindIII, HpaI, MspI, EcoRI, and SstI restriction endonucleases, respectively. In each case, hybridizing fragments from analbuminemic and control DNA are the same length. The decreased intensity of hybridization of the major HindIII DNA fragment from the analbt~inemic genome (2A-) results from inccc~plete digestion of this DNA. We have verified this in other experiments (not shown). The size markers are from coelectrophoresis of EcoRI digested XDNA and from HaeIII digested ~ X 174 DNA.
electrophoresis (not shown), and may not be normal albumJm sensu stricto. may also be pertinent to add that we found no conpensatorv(2~fetoprotein
It (
ng/ml) in our patient by radioirsmlnoassay. In contrast to the profound phenotypic difference, seen in Fig. I, no differences between the pattern of hybridizing DNA fragments from the analbu~tinemic gene and that from a normal gene could be observed (Fig. 2). The sizes of these fragments are in close agreement with those expected from the mapping of t/he human serum albumin gene previously determined in this laboratory (4). We, therefore, conclude that the him/an sert~n albumin gene is not only present in congenital analbuminemia but that it is grossly identical structurally to the normal gene.
Furthermore, the similar in%~ensities of the
819
Vol. 116, No. 3, 1983
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
hybridizing fragments indicate that there is tJ%e same number of copies of this gene in beth the a/qalbtmttne/~ic and the normal genome.
Actually, we have
previously concluded that the albumin gene is unique in the human genome (4). From the present study we cannot e~xclude, however, the existence of ~mall rearrangements or deletions within the gene or in its flanking regions.
DISCT]SSION The technique of studying the gross rearrangement of a gene by restriction endonuclease cleavage and Southern hybridization analysis has successfully detected gene deletions as a cause for the lack of human prote~ms.
Deletions related to the thalasemias (13) or the deletion of the
human chorionic s o m a t ~ t r o p i n
gene (14) are similar in that they occurred
within clusters of related genes located over a small region on the same chromosome.
Deletion of an entire gene within such a cluster could have
arisen by unequal crossing over between similar sequences of related genes. The only gene known to be linked (5,15) and related (16-19) to albtmtin is the (~-fetoprotein gene.
However, since this study revealed no gross
deletions of the albumin gene, a somewhat different mechanism must be responsible for the albumin deficiency.
Increased degradation of normal
serum albumin is not the cause as indicated by turnover studies of
131I-labelled albumin injected into amalbumine~tic patients.
In fact,
these patients have significantly longer albumin survival times than normal individuals f20). Esumi et at (21) have shown t/qat the disorder in a stra~]~ of ap~%lbuminemic rats appears to be due to a mutation affecting the maturation of albumin mRNA.
They also detected no gross rearrangement .in the gene for
rat albumin (21), but subsequent cloning and sequencing in the analbuminemic region revealed a seven base pair deletion in one intron (22). Our data in man are consistent with such a model.
Further fine structural analvsis of
the analbuminemic gene and its flanking regions are planned to determine tile cause of this disorder.
820
VOI. 116, No. 3, 1 9 8 3
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS RF/~RENCES
i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15. 16. ]7. 18. 19. 20. 21. 22.
Rothschild, M.A. and Oratz, M. (1976) Structure and function of plasma proteins, pp. 79-105, Ed: Allison AC, Plenum, New York. Russi, E. and Weigand, K. (1983) Klin. Wschr. in press. Dugaiczyk, A., Law, S.W., and Dennison, O.E. (1982) Proc. Natl. Acad. Sci. USA 79:71-75; 2124. Hawkins, J.W. and Dugaiczyk, A. (1982) Gene, 19,55-58. Harper, M.E. and Dugaiczyk, A. (1983) Am. J. Hum. C~net. in press. Weigand, K., Russi, E., von Schulthess, G., and Bavaud, C. (1983) Klin. Wschr. in press. Goosens, M. and Kan, W.Y. (1981) Methods in F~lzymology, 76, 805-817. Kan, Y.W., Dozy, A.M., Trecartin, R., and Todd, D. (1977) N. Engl. J. Med. 297, 1081-1084. Hel!ing, R.B., Goodman, H.M., and Boyer, H.W. (1974) J. Virol. 14, 1235-1244. Southern, E.M. (1975) J. Mol. Biol. 98, 503-517. Maniatis, T., Jeffrey, A., and Kleid, D.G. (1975) Proc. Natl. Acad. Sci. USA, 72, 1184-1188. Denhardt, D.T. (1966) Biochem. Biophys. Res. ComTmn. 23, 641-646. Meats, J.G., Ramirez, F., I~ibowitz, D., Nakamura, F., Bloom, A., Konotey-Ahulu, F., and Bank, A. (1978) Proc. Natl. Acad. Sci. USA 75, 1222-1226. Wurzel, J.M., Parks, J.S., Herd, J.E., and Nielsen, P.V. (1982) DNA I, 251-257. D'Eustachio, P., Ingrain, R.S., Tilghman, S.M., and Ruddle, F.H. (1981) Sonmmtic Cell Genet. 7, 289-294. Jagodzinski, L.L., Sargent, T.D., Yang, M., Glackin, C., and Bonner, J. (1981) Proc. Natl. Acad. Sci. USA 78, 3521-3525. Law, S.W., and Dugaiczyk, A. (1981) Nature 291, 201-205. Gorin, M.B., Cooper, D.L., Eiferman, F., van de Rijn, P., and Tilghman, S.M. (1981) J. Biol. Chem. 256, 1954-1959. Beattie, W.G., and Dugaiczyk, A. (1982) Gene 20, 415-422. Waldman, T. (1977) F~. Rosenver V, Oratz M, Rothschild MA, Oxford, Pergamon pp. 263-266. Esumi, H., Takahashi, Y., Sekiya, T., Sato, S., Nagase, S., and Sugimira, T. (1982) Proc. Natl. Acad. Sci. USA 79, 734-738. Esumi, H., Takahashi, Y., Sato, S., Nagase, S., and Sugimura, T. (1983) Proc. Natl. Acad. Sci. USA 80, 95-99.
821
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pages 817-821
November 15, 1983
STRUCIUPAL INTF~PIq~f OF ~ H79iAN ALBUMIN G ~ IN CONGEb~TAL 7~A]NB[~IIK~IA
Robert A. Avery.1 , Elliot Alpert 2 , Kurt ~ i g a n d 3 , and Achilles Dugaiczyk I'4 I)
Department of Cell Biology, Bavlor College of Medicine, Houston, Texas 77030 2) C~stroenterologyDivision, Department of Medicine, Baylor College of Medicine, Houstmn, T~xas 77030 3) Department of Clinical Pharmacology, [~iversity of Berne, Berne,~tzerland 4) Department of Biochemist~,, University of California, Riverside, California 92521 Received September 9, 1983 The human serum albumin gene was analyzed by restriction endonuclease mapping of chromosomal DNA isolated from a patient with congenital analbuminemia. Following digestion with a variety of restriction e_ndonucleases, tJne DNA from this individual produced the same fragments with homology to a serum albumin cDNA probe as did a control DNA specimen. Therefore, the genetic condition of congenital ana]buminemia is not caused by any gross structural rearrange]nent or deletion of the gene itself, but may result from an abnormality in the gene's fine structure, perhaps affece~ing regulation or processing of the primary RNA transcript.
Alb%mtin normally accounts for about 60 percent of the total serum proteins and helps to maintain osmotic pressure, and to transport ions, fatty acids, metabolites, hormones, and drugs (1). However, in the rare genetically transmitted disorder of analbt~ninemia, only trace amounts of this protein are detectable in the serum.
Despite albumin's varied functions, its
virtua]Iv complete absence produces surprisingly few ~%ynlotoms. Edema is present in most of the 22 reported cases, but other serious con~lications are not present.
The increased level of other plasma proteins which usually
acco~oanies analbuminemia partially compensates for the lack of albumin, alleviating the consequences of its absence (2). In an effort to characterize the molecular nature of this genetic abnormality, we have compared the gross structure of the serum albumin gene Send correspondence to: Dr. Elliot Alpert, Gastroenterology Section, Department of Medicine, 6565 Fannin, B-503 , Houston, Texas 77030
0006-291 X/83 $1.50 817
Copyright © 1983 by Academic Press, Inc. All rights o f reproduction in any form reserved.
Vol. 116, No. 3, 1 9 8 3
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
from an analbuminemic and control individual by restriction endonuclease analysis.
This study has been made possible by isolation of DNA from blood
of a newly discovered case of analbumine/nia, and bs~ the availability of two human serum albuntin cDNA clones which were constructed and sequenced in this laboratory (3). These two overlapping clones, pHA36 and pH/k2.06, encode 2.1 kb of hums~m serum albumin mRkih seq~lence fro~ the 3' poly-(A) tail to within 62 bases of the CAP site.
The entire amino acid sequence of albumin is
encoded on this 2.1 P~ stretch of cD~iA, which in turn is d i s ~ r s e d into multiple exons over a region of 16 kb of chromosomal D}IA (4) located on qli-22 of human chromosome 4 (5).
MATERTALS AND ~ T H O D S Blood was obtained from a 60 year old Swiss nmn (6). }Iigh molecular weight D~IA was isolated from peripheral blood ]eukocytes by tJne method of Goosens and Kan (7) , except that the red blood cells were eliminated ~! lysis with ammonium chloride (8) befre the white blood cells were digested with proteinase K. TweDty ~g aliquots of this analbuminemic DNA and of normal htmmn DNA were digested to conpletion with AvaII, HindIII, HpaI, MspI, EcoRI, and SstI restriction endonucleases according to the suppliers' (New England Biolabs and Bethesda Research Ix~boratories) instructions. The resulting fragments were separated by electrophoresis through a 1% agarose gel (9) and transferred to a nitrocellulose paper (Schleicher & Schuell, BAd5) by Sou%/nern' s procedure (I0). The probes, ~ 6 stud pHA206, were nick-translated (Ii) to a sDecific activity of I0- [~ p] cpm/~g DNA and then hybridized to the DNA-containing nitrocellulose paper in a solution containing 0.2% SDS, 5xSSPE (SSPE = 0.18 M NaCl, 10ram sodium phosphate, 1ram ~IYPA; pH 7.0) and 5x Demhardt's solution (12) for 18 hrs at 68°C. After the filter was washed at 68°C with 2xSSPE and dried, it was autoradiographed wit/n Dupond Cronex film and an intensifying screen. RESULTS Fig. 1 shows the electrophoretic separation of serum proteins from a control and the analbuminemic individual analyzed in the present study. There is a striking absence of any protein in the region of the most prominent albumin band from the control serum.
However, it may be
interesting to note that a trace amount of albumin (1.7 rag/100 ml; normal concentration:
4.2
g/100 ml) was found (6) in our analbuminemic patient by
specific inrmlnoprecipitation,
but it had a slightly different migration
velocity than normal albumin when subjected to polyacrvlamide gel
818
Vol. 116, No. 3, 1 9 8 3
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS 1 2 3 4 5 6 N A - N A - N A - N A - N A-N A-
i
2
Kb
®
® Fig. 1
Cellulose acetate electrophoretic separation of serum proteins from a control (lane I) and the analbuminemic serum (lane 2) used in the present study.
Fig. 2
Autoradiogram of_restriction endonucl~@se digested normal (N) and analbtmd_nemic (A) DNA hybridized to --P-labelled human albtmlin cDNA clones pHA36 and pHA206 (3). The DNA in the two lanes under heading i have been digested with AvaII. Similarly, headings 2,3,4,5, and 6, correspond to diges-~{ons with HindIII, HpaI, MspI, EcoRI, and SstI restriction endonucleases, respectively. In each case, hybridizing fragments from analbuminemic and control DNA are the same length. The decreased intensity of hybridization of the major HindIII DNA fragment from the analbt~inemic genome (2A-) results from inccc~plete digestion of this DNA. We have verified this in other experiments (not shown). The size markers are from coelectrophoresis of EcoRI digested XDNA and from HaeIII digested ~ X 174 DNA.
electrophoresis (not shown), and may not be normal albumJm sensu stricto. may also be pertinent to add that we found no conpensatorv(2~fetoprotein
It (
ng/ml) in our patient by radioirsmlnoassay. In contrast to the profound phenotypic difference, seen in Fig. I, no differences between the pattern of hybridizing DNA fragments from the analbu~tinemic gene and that from a normal gene could be observed (Fig. 2). The sizes of these fragments are in close agreement with those expected from the mapping of t/he human serum albumin gene previously determined in this laboratory (4). We, therefore, conclude that the him/an sert~n albumin gene is not only present in congenital analbuminemia but that it is grossly identical structurally to the normal gene.
Furthermore, the similar in%~ensities of the
819
Vol. 116, No. 3, 1983
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
hybridizing fragments indicate that there is tJ%e same number of copies of this gene in beth the a/qalbtmttne/~ic and the normal genome.
Actually, we have
previously concluded that the albumin gene is unique in the human genome (4). From the present study we cannot e~xclude, however, the existence of ~mall rearrangements or deletions within the gene or in its flanking regions.
DISCT]SSION The technique of studying the gross rearrangement of a gene by restriction endonuclease cleavage and Southern hybridization analysis has successfully detected gene deletions as a cause for the lack of human prote~ms.
Deletions related to the thalasemias (13) or the deletion of the
human chorionic s o m a t ~ t r o p i n
gene (14) are similar in that they occurred
within clusters of related genes located over a small region on the same chromosome.
Deletion of an entire gene within such a cluster could have
arisen by unequal crossing over between similar sequences of related genes. The only gene known to be linked (5,15) and related (16-19) to albtmtin is the (~-fetoprotein gene.
However, since this study revealed no gross
deletions of the albumin gene, a somewhat different mechanism must be responsible for the albumin deficiency.
Increased degradation of normal
serum albumin is not the cause as indicated by turnover studies of
131I-labelled albumin injected into amalbumine~tic patients.
In fact,
these patients have significantly longer albumin survival times than normal individuals f20). Esumi et at (21) have shown t/qat the disorder in a stra~]~ of ap~%lbuminemic rats appears to be due to a mutation affecting the maturation of albumin mRNA.
They also detected no gross rearrangement .in the gene for
rat albumin (21), but subsequent cloning and sequencing in the analbuminemic region revealed a seven base pair deletion in one intron (22). Our data in man are consistent with such a model.
Further fine structural analvsis of
the analbuminemic gene and its flanking regions are planned to determine tile cause of this disorder.
820
VOI. 116, No. 3, 1 9 8 3
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS RF/~RENCES
i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15. 16. ]7. 18. 19. 20. 21. 22.
Rothschild, M.A. and Oratz, M. (1976) Structure and function of plasma proteins, pp. 79-105, Ed: Allison AC, Plenum, New York. Russi, E. and Weigand, K. (1983) Klin. Wschr. in press. Dugaiczyk, A., Law, S.W., and Dennison, O.E. (1982) Proc. Natl. Acad. Sci. USA 79:71-75; 2124. Hawkins, J.W. and Dugaiczyk, A. (1982) Gene, 19,55-58. Harper, M.E. and Dugaiczyk, A. (1983) Am. J. Hum. C~net. in press. Weigand, K., Russi, E., von Schulthess, G., and Bavaud, C. (1983) Klin. Wschr. in press. Goosens, M. and Kan, W.Y. (1981) Methods in F~lzymology, 76, 805-817. Kan, Y.W., Dozy, A.M., Trecartin, R., and Todd, D. (1977) N. Engl. J. Med. 297, 1081-1084. Hel!ing, R.B., Goodman, H.M., and Boyer, H.W. (1974) J. Virol. 14, 1235-1244. Southern, E.M. (1975) J. Mol. Biol. 98, 503-517. Maniatis, T., Jeffrey, A., and Kleid, D.G. (1975) Proc. Natl. Acad. Sci. USA, 72, 1184-1188. Denhardt, D.T. (1966) Biochem. Biophys. Res. ComTmn. 23, 641-646. Meats, J.G., Ramirez, F., I~ibowitz, D., Nakamura, F., Bloom, A., Konotey-Ahulu, F., and Bank, A. (1978) Proc. Natl. Acad. Sci. USA 75, 1222-1226. Wurzel, J.M., Parks, J.S., Herd, J.E., and Nielsen, P.V. (1982) DNA I, 251-257. D'Eustachio, P., Ingrain, R.S., Tilghman, S.M., and Ruddle, F.H. (1981) Sonmmtic Cell Genet. 7, 289-294. Jagodzinski, L.L., Sargent, T.D., Yang, M., Glackin, C., and Bonner, J. (1981) Proc. Natl. Acad. Sci. USA 78, 3521-3525. Law, S.W., and Dugaiczyk, A. (1981) Nature 291, 201-205. Gorin, M.B., Cooper, D.L., Eiferman, F., van de Rijn, P., and Tilghman, S.M. (1981) J. Biol. Chem. 256, 1954-1959. Beattie, W.G., and Dugaiczyk, A. (1982) Gene 20, 415-422. Waldman, T. (1977) F~. Rosenver V, Oratz M, Rothschild MA, Oxford, Pergamon pp. 263-266. Esumi, H., Takahashi, Y., Sekiya, T., Sato, S., Nagase, S., and Sugimira, T. (1982) Proc. Natl. Acad. Sci. USA 79, 734-738. Esumi, H., Takahashi, Y., Sato, S., Nagase, S., and Sugimura, T. (1983) Proc. Natl. Acad. Sci. USA 80, 95-99.
821