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Isoelectric focusing of serum in cystic fibrosis: Failure to distinguish between homozygote and heterozygote sera
459
Clinica Chimica Acta, 70 (1976) 459-462 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
SHORT COMMUNICATION __-
___-
___~__
CGA 7909
ISOELECTRIC FOCUSING OF SERUM IN CYSTIC FIBROSIS: FAILURE TO DISTINGUISH BETWEEN HOMOZYGOTE AND HETEROZYGOTE SERA
GORDON FORSTNER *, ALY KOHEIL and JANET FORSTNER Kinsmen Avenue,
Cystic Fibrosis Research Centre, The Hospital Toronto, Ontario M5G 1X8 (Canada)
for Sick Children,
555
University
(Received March 2,1976)
Summary Thin-layer isoelectric focusing was performed on samples of sera from patients with Cystic Fibrosis, siblings and obligate heterozygotes (parents), and children without cystic fibrosis (controls). The protein band with an isoelectric point of pH 5.48, previously reported to be absent in homozygote cystic fibrosis sera, was found to have a pl of 5.25. It was present in approximately one-half of the homozygote and heterozygote sera tested, and absent from 18 percent of control sera. The presence of this band is not therefore a reliable marker for the normal gene, and cannot be used to identify the heterozygous carrier for cystic fibrosis.
The diagnosis of the heterozygous carrier of the cystic fibrosis gene is far from satisfactory. The heterozygote has higher sweat electrolytes than normal controls [I], and possesses serum which contains the ciliary dyskinesia factor [ 2,3,4]. However, neither of these parameters can be used to distinguish him (or her) reliably from the homozygote [ 1,4]. In 1973, Wilson et al. [5], using isoelectric focusing of serum, provided evidence to suggest that this technique could identify not only the serum factorgamma globulin complex [ 31, with an alkaline pl of 8.41, but also a second protein with alower pl of 5.48 which appeared to be a marker for the ncrmal gene. If this observation were correct, the heterozygote could be identified relatively simply as an individual with both the serum factor and the protein with the pZ of 5.48. The latter protein is also of substantial interest because it could repre* To
whom
all correspondrnce
should
be addressed.
460
sent a deletion causally related to the disease. Wilson and Fuden berg [6 3 have suggested, for example, that it might represent a fragment of ff~-macroglobu~in which disappears from C/F serum as the result of diminished proteolytic activity. For these reasons we sought to confirm the results claimed for the pI 5.48 protein as a marker for the normal gene. Patients with a diagnosis of cystic fibrosis, established by clinical history, and high sweat chloride levels (over 60 mequiv./l), parents, siblings, and a variety of patients without family history or evidence of cystic fibrosis, were studied. Thin-layer isoelectric focusing was performed on an LKB-Multiphor App~atus at 4°C using conditions which were similar to those of Wilson et al. [ 51, except for minor modifications which improved the resolution of bands in the pH 5.0 to 6.0 zone. A gel containing 2% (w/v) ampholine carrier amphobytes, pH 5-7, 4 M urea and 5% (T) acrylamide (C = 3%) was used. Polymerization was accomplished with riboflavin 0.04%, and accelerated by addition of 0.8 ml of 1% ammonium persulfate (25 mg per plate). Gels were stored over-
CFH
HH
Fig. 1. Thin-layer isnelectrie focusing with ampholinr carrier gradient PH 5.7 of sera from Patient with cystic fibrosis (C/F), and sew from three heterozygotes (H). 10 ~1 was applied to the gel in each case. a = band with pl of 5.25 i 0.02, h = reference band with p7 of 5.23 i 0.01.
461
TABLE I PRESENCE OF pl 5.25 BAND AND CONTROL SERUM
IN CYSTIC
FIBROSIS
HOMOZYGOTE,
HETEROZYGOTE.
SIBLING
The age of the groups is presented as mean +_S.E.; in parentheses the range is given. Test group
Cystic Fibrosis Homozygotes Heterozygotes (parents) Siblings
NO.
26 35 15
Controls Total
27
Males
10
Females
17
Age (year) *
PI
5.25 band
Present
Absent
% present
11.7 t 1.2 (3-25) 38.3 + 13.0 (24-59) 12.1 f 5.0 (5-19)
14
12
54
18
17
51
8
7
53
9.63 i 0.90 (2-17) 8.80 + 1.55 (2-17) 10.71 f 0.69 (3-17)
22
5
a2
9
1
90
13
4
76
night at 4°C and focused at 400 V for 1 h prior to sample application. lo-30 ~1 of sample was applied to Whatman 3 MM paper squares 0.5 cm below the cathode, and focusing continued at 400 V for 18 h, followed by 800 V for 4 h. The gels were stained in 0.2% w/v Coomassie blue in ethanol/water/acetic acid (25 : 63 : 8, v/v/v). To obtain serum for isoelectric focusing, samples of blood were taken from patients who had fasted overnight. The blood was clotted at 4°C for 4-6 hours, and centrifuged at 1500 X g for 30 min to separate serum. Sera were divided into O.l-ml aliquots and stored at -20°C until used. All isoelectric focusing was performed within 14 days of serum collection. After staining, the gels were read by one of us (G.F.) who was unaware of the source of the individual sera. Scoring was not correlated with the identity of the sera until the end of the study. If the band was not observed or was equivocal with 10 ~1 of serum, the procedure was repeated using 30 ~1 serum. Fig. 1 illustrates a typical isoelectric focusing run. Our results differ from those published [5] in that the band a, previously designated as having a pl of pH 5.48 was found to have a pl of 5.25 + 0.02 [6]. The identity of the band is unmistakable however because of its proximity to reference band b (pl 5.23 + 0.01 [6]), which is prominently seen in all sera. Notable in this run is the fact that the pl 5.25 band is present in both heterozygous and homozygous cystic fibrosis sera. Cumulative results are shown in Table I. Of 26 patients with cystic fibrosis, 14 had a protein band with pZ 5.25. In parents and siblings a similar frequency was observed. In 27 control patients the band appeared more frequently, particularly amongst males. The volume of serum required for demonstration was not significantly higher in heterozygotes or homozygotes for the C/F trait than normal controls. No other relation to age, sex, or clinical state was discernible.
Although these results suggest that the ~15.25 band occurs more commonly in sera from normal controls, they clearly indicate that the protein is not an invariable deletion in cystic fibrosis nor is it always found in the controls. Similar results, at variance with those of Wilson et al. [6]) appear to have been obtained by AltIand et al. [ 71. Detection of the ~15.25 band is therefore of no use in defining the heterozygote. Our results do suggest however that the pl 5.25 protein is more common in persons without the C/F gene, and therefore it is possible that its absence from some C/F sera may distantly reflect a basic abnormality in the disease. References 1 2 3 4 5 6 7
Sproul, A. and Hueng, N. (1966) J. Pediatr. 69,759-770 Speck, A., Heick, H., Cress, H. and Logan, W. (1967) Pediatr. Res. I, 173-177 Schmoyer, I., Fischer, J. and Brooks, S, (1972) Biachem. Biophys. Res. Commun. 46, 1923-1927 Forstner, G.. Crazier. D. and Sturgess, J. (1975) Can. Med. Assoc. J. 113, 550-556 Wilson, G., Jahn, T. and Fonseca, J. (19’73) Clin. C&n. Acta 49, 79-91 Wilson, G. and Fudenberg, H. (1976) Pediatr. Res. 10, 87-6 Altland, K., Schmidt, S., Kaiser, G. and Knoche, W. (1975) Humangenetik 28, 207-216
Clinica Chimica Acta, 70 (1976) 459-462 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
SHORT COMMUNICATION __-
___-
___~__
CGA 7909
ISOELECTRIC FOCUSING OF SERUM IN CYSTIC FIBROSIS: FAILURE TO DISTINGUISH BETWEEN HOMOZYGOTE AND HETEROZYGOTE SERA
GORDON FORSTNER *, ALY KOHEIL and JANET FORSTNER Kinsmen Avenue,
Cystic Fibrosis Research Centre, The Hospital Toronto, Ontario M5G 1X8 (Canada)
for Sick Children,
555
University
(Received March 2,1976)
Summary Thin-layer isoelectric focusing was performed on samples of sera from patients with Cystic Fibrosis, siblings and obligate heterozygotes (parents), and children without cystic fibrosis (controls). The protein band with an isoelectric point of pH 5.48, previously reported to be absent in homozygote cystic fibrosis sera, was found to have a pl of 5.25. It was present in approximately one-half of the homozygote and heterozygote sera tested, and absent from 18 percent of control sera. The presence of this band is not therefore a reliable marker for the normal gene, and cannot be used to identify the heterozygous carrier for cystic fibrosis.
The diagnosis of the heterozygous carrier of the cystic fibrosis gene is far from satisfactory. The heterozygote has higher sweat electrolytes than normal controls [I], and possesses serum which contains the ciliary dyskinesia factor [ 2,3,4]. However, neither of these parameters can be used to distinguish him (or her) reliably from the homozygote [ 1,4]. In 1973, Wilson et al. [5], using isoelectric focusing of serum, provided evidence to suggest that this technique could identify not only the serum factorgamma globulin complex [ 31, with an alkaline pl of 8.41, but also a second protein with alower pl of 5.48 which appeared to be a marker for the ncrmal gene. If this observation were correct, the heterozygote could be identified relatively simply as an individual with both the serum factor and the protein with the pZ of 5.48. The latter protein is also of substantial interest because it could repre* To
whom
all correspondrnce
should
be addressed.
460
sent a deletion causally related to the disease. Wilson and Fuden berg [6 3 have suggested, for example, that it might represent a fragment of ff~-macroglobu~in which disappears from C/F serum as the result of diminished proteolytic activity. For these reasons we sought to confirm the results claimed for the pI 5.48 protein as a marker for the normal gene. Patients with a diagnosis of cystic fibrosis, established by clinical history, and high sweat chloride levels (over 60 mequiv./l), parents, siblings, and a variety of patients without family history or evidence of cystic fibrosis, were studied. Thin-layer isoelectric focusing was performed on an LKB-Multiphor App~atus at 4°C using conditions which were similar to those of Wilson et al. [ 51, except for minor modifications which improved the resolution of bands in the pH 5.0 to 6.0 zone. A gel containing 2% (w/v) ampholine carrier amphobytes, pH 5-7, 4 M urea and 5% (T) acrylamide (C = 3%) was used. Polymerization was accomplished with riboflavin 0.04%, and accelerated by addition of 0.8 ml of 1% ammonium persulfate (25 mg per plate). Gels were stored over-
CFH
HH
Fig. 1. Thin-layer isnelectrie focusing with ampholinr carrier gradient PH 5.7 of sera from Patient with cystic fibrosis (C/F), and sew from three heterozygotes (H). 10 ~1 was applied to the gel in each case. a = band with pl of 5.25 i 0.02, h = reference band with p7 of 5.23 i 0.01.
461
TABLE I PRESENCE OF pl 5.25 BAND AND CONTROL SERUM
IN CYSTIC
FIBROSIS
HOMOZYGOTE,
HETEROZYGOTE.
SIBLING
The age of the groups is presented as mean +_S.E.; in parentheses the range is given. Test group
Cystic Fibrosis Homozygotes Heterozygotes (parents) Siblings
NO.
26 35 15
Controls Total
27
Males
10
Females
17
Age (year) *
PI
5.25 band
Present
Absent
% present
11.7 t 1.2 (3-25) 38.3 + 13.0 (24-59) 12.1 f 5.0 (5-19)
14
12
54
18
17
51
8
7
53
9.63 i 0.90 (2-17) 8.80 + 1.55 (2-17) 10.71 f 0.69 (3-17)
22
5
a2
9
1
90
13
4
76
night at 4°C and focused at 400 V for 1 h prior to sample application. lo-30 ~1 of sample was applied to Whatman 3 MM paper squares 0.5 cm below the cathode, and focusing continued at 400 V for 18 h, followed by 800 V for 4 h. The gels were stained in 0.2% w/v Coomassie blue in ethanol/water/acetic acid (25 : 63 : 8, v/v/v). To obtain serum for isoelectric focusing, samples of blood were taken from patients who had fasted overnight. The blood was clotted at 4°C for 4-6 hours, and centrifuged at 1500 X g for 30 min to separate serum. Sera were divided into O.l-ml aliquots and stored at -20°C until used. All isoelectric focusing was performed within 14 days of serum collection. After staining, the gels were read by one of us (G.F.) who was unaware of the source of the individual sera. Scoring was not correlated with the identity of the sera until the end of the study. If the band was not observed or was equivocal with 10 ~1 of serum, the procedure was repeated using 30 ~1 serum. Fig. 1 illustrates a typical isoelectric focusing run. Our results differ from those published [5] in that the band a, previously designated as having a pl of pH 5.48 was found to have a pl of 5.25 + 0.02 [6]. The identity of the band is unmistakable however because of its proximity to reference band b (pl 5.23 + 0.01 [6]), which is prominently seen in all sera. Notable in this run is the fact that the pl 5.25 band is present in both heterozygous and homozygous cystic fibrosis sera. Cumulative results are shown in Table I. Of 26 patients with cystic fibrosis, 14 had a protein band with pZ 5.25. In parents and siblings a similar frequency was observed. In 27 control patients the band appeared more frequently, particularly amongst males. The volume of serum required for demonstration was not significantly higher in heterozygotes or homozygotes for the C/F trait than normal controls. No other relation to age, sex, or clinical state was discernible.
Although these results suggest that the ~15.25 band occurs more commonly in sera from normal controls, they clearly indicate that the protein is not an invariable deletion in cystic fibrosis nor is it always found in the controls. Similar results, at variance with those of Wilson et al. [6]) appear to have been obtained by AltIand et al. [ 71. Detection of the ~15.25 band is therefore of no use in defining the heterozygote. Our results do suggest however that the pl 5.25 protein is more common in persons without the C/F gene, and therefore it is possible that its absence from some C/F sera may distantly reflect a basic abnormality in the disease. References 1 2 3 4 5 6 7
Sproul, A. and Hueng, N. (1966) J. Pediatr. 69,759-770 Speck, A., Heick, H., Cress, H. and Logan, W. (1967) Pediatr. Res. I, 173-177 Schmoyer, I., Fischer, J. and Brooks, S, (1972) Biachem. Biophys. Res. Commun. 46, 1923-1927 Forstner, G.. Crazier. D. and Sturgess, J. (1975) Can. Med. Assoc. J. 113, 550-556 Wilson, G., Jahn, T. and Fonseca, J. (19’73) Clin. C&n. Acta 49, 79-91 Wilson, G. and Fudenberg, H. (1976) Pediatr. Res. 10, 87-6 Altland, K., Schmidt, S., Kaiser, G. and Knoche, W. (1975) Humangenetik 28, 207-216