Vol. 45, No. 4, July 1998
BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Pages643-650
AMPLIFICATION OF PHENYLALANINE HYDROXYLASE AND CYSTATHIONINE ~-SYNTHASE TRANSCRIPTS IN HUMAN PERIPHERAL LYMPHOCYTES BY RT-PCR K. SANTHI DEVI 1 , A. RADHA RAMA DEVI 2 AND PATURU KONDAIAH 1. 1.Department of Molecular Reproduction, Development and Genetics and 2. Health Centre, Indian Institute of Science, Bangalore - 560 012, INDIA Received March20, 1998 Received after revision,March 23, 1998
Summary A simple, rapid, reliable and convenient method was developed to analyze the gene defects in Phenylketonuria (PKU) and Homocystinuria (HCU). In this method, illegitimately transcribed phenylalanine hydroxylase (PAH) and cystathionine 13-synthase (CBS) mRNAs in peripheral lymphocytes were used as templates for amplification by RT-PCR. The amplified products were confirmed by restriction enzyme digestions, southern blot hybridizations and sequencing. Point mutations in the protein coding region and splice junction mutations of PAH and CBS can be analyzed by this method. KEY WORDS: Illegitimate transcription, Phenylalanine hydroxylase, Phenylketonuria, Cystathionine [3synthase, Homocystinuria, Reverse Transcription, Polymerase Chain Reaction, Mental retardation
INTRODUCTION Analysis of the molecular lesions in genetic disorders has been the recent focus for prenatal diagnosis, genetic counseling and population screening.
At the molecular level, a
disease can manifest due to deletions and duplications of the coding regions, splicing defects of the RNA and point mutations etc. Several methods have been developed to diagnose molecular defects, which include RFLP analysis by southern blot, PCR amplifications of exonic regions from genomic DNA followed by sequence analysis, Chemical cleavage of mismatch, PCR followed by SSCP analysis to detect point mutations and RT-PCR amplification from RNA isolated from the respective cells or tissue for direct sequence analysis.
Address for correspondance * E-mail :
[email protected] 1039-9712/98/100643-08505.0010 643
Copyright 9 1998 by Academic Press Australia. All rights of reproduction in any form reserved.
Vol. 45, No. 4, 1998
BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL
Amplification of ectopic/leaky/illegitimately transcribed mRNA
and subsequent
sequence analysis has great advantage over the above mentioned approaches.
Illegitimate
transcription is the low level of transcription of tissue specific genes outside of the tissue where they are normally active (1,2). A number of point mutations have been identified by using this technique in many diseases namely, factor VIII (3) dystrophin gene (4,5), Cystic fibrosis transmembrane conductance regulator gene (6,7,8), Phenylketonuria and Homocystinuria (9,10).
Phenylalanine hydroxylase is a hepatic enzyme that catalyzes the irreversible conversion of phenylalanine to tyrosine.
PKU, one of the commonest inborn errors of amino acid
metabolism is an autosomal recessive disease, caused by a deficiency of the hepatic enzyme phenylalanine hydroxylase. A full-length human phenylalanine hydroxylase eDNA of 2449 bp has been reported from a liver library (11). In this sequence, the first methionine codon is at nucleotide position 223 followed by an open reading frame of 1353 bp encoding 451 amino acids.
The gene has been mapped to chromosome 12 q 24.1. This gene is about 90 Kb and
contains 13 exons. The intron sizes range from 1 Kb to 23.5 Kb (12).
Homocystinuria is another common autosomal recessive metabolic disease associated with vascular occlusions, osteoporosis, skeletal abnormalities, dislocated optic lenses and mental retardation (MR). HCU is mainly due to a total or partial deficiency of cystathionine [3-synthase, which catalyzes the first irreversible step of homocysteine transsulfuration. It is a pyridoxal 5' phosphate (PLP) dependent enzyme which condenses homocysteine and serine to form cystathionine. The human CBS eDNA has been isolated from liver fibroblasts (13) and a human hepatoma cell line, HepG2 (14). The reported sequence from the liver was 2554 nucleotides encoding the CBS subunit of 551 amino acids (13). The CBS gene is at least 23 kb long with 17 exons and maps to chromosome 21 q 22.3.
Several mutations in PAH and CBS genes have been reported using exon amplification, RT-PCR of mRNA isolated from liver, fibroblasts and EBV transformed lymphocytes (9,10). However, in order to minimize the time and cost, we developed an easy method to amplify and sequence the coding regions of both PAH and CBS RNA's by RT-PCR Of illegitimate transcripts directly from lymphocyte RNA with out the need for fibroblast cultures and EBV-transformation of the lymphocytes.
644
Vol. 45, No. 4, 1998
MATERIALS
BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL
AND METHOD
Isolation of lymphocytes, RNA extraction and cDNA synthesis: Lymphocytes from 10 ml venous blood were isolated by density centrifugation on Ficoll Paque (Pharmacia Biotech, Sweden) and washed thrice with 1X Phosphate buffered saline. Total RNA was extracted from 1 X107 cells using RNAesy colmnn (Qiagen GmbH, Germany) as per the manufacturers protocol. The total RNA from a human hepatoma cell line (HepG2) used as positive control was isolated by a single step purification method (15). The RNAs were quantitated and the integrity was assessed using denaturing agarose gels containing 2.2 M formaldehyde. One ~tg of total RNA along with oligo dT or random hexamers heated to 70~ for 10 min. was used for cDNA synthesis. The annealed RNA was incubated with Reverse transcriptase (MMLV RT, Promega, USA) along with rest of the components at 37~ for 1 hr. The reaction was stopped by inactivating the enzyme at 70~ for 15 min. and treated with E.coli RNase H (0.8 units) for 30 min. at 37~ 30~tl water was added to the reaction mixture and passed through a Sephadex G-50 spun column equilibrated with 10 mM Tris/1 mM EDTA (pH 8.0). A portion of the cDNA (100 ng equivalent of total RNA) was used for PCR amplifications.
PCR amplifications cDNA from the earlier step was subjected to first round PCR amplification using the relevant primer sets. Typical cycling parameters were, denaturation at 94~ for 1 min., annealing at 60~ for 1 min., primer extension at 72~ for 3 min. for 35 cycles followed by final extension at 72~ for 8 min. in a thermal cycler (MJ Research, USA). For the second round (nested PCR), one tenth of the first round PCR product was used along with the internal primer sets. The sequences of the primers and their relative positions on the cDNA are listed in Table-1. The primer sets, expected size of the products, enzyme cleavage sites and the size of the digested fragments are listed in the Table -2. A portion of the amplified products was electrophoresed in 2% agarose gel containing ethidium bromide and the DNA was visualized using an UV transilluminator.
Sequencing of the DNA fragments: For sequencing, the PCR products were resolved on a 2% agarose gel and the fragments of interest were purified by gel extraction columns (Qiagen GmbH, Germany). The purified DNA fragments were sequenced in an automated sequencer (Applied Biosystems Inc., USA). The sequence data was analyzed using DNA Star software. Amplifications using the RNA isolated from HepG2 cell line served as a positive control for both the genes.
RESULTS AND DISCUSSION Mutation analysis of single gene disorders requires a rapid, reliable and less expensive technique. This is more so for prenatal diagnosis where rapid analysis is called for and in population screening, which require handling of large sample size. We have developed a method
645
Vol. 45, No. 4, 1998
BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL
T a b l e -1 Relative location on the liver c D N A
Sequence
Pahl
166-189
5' AACCTGCCTGTACGTGAGGCCCTA 3'
Pah2
1066-1086"
5' CAACAGCTCATGGCAGATGTC 3'
Pah3
907-927
5' GACGTTTCTCAATTCCTGCAG 3'
Pah4
1594-1617"
5' GATTCACAGCTGACAGACCACATT 3'
Cbsl
94-114
5' CCAGGATCCCATGACAGATTC 3'
Cbs2
738-758"
5' TGGGCGTCCTCACAATCTCAG 3'
Cbs3
654-673
5' CGGCAGTGAGGGGCTATCGCT 3'
Cbs4
1254-1274"
5' GCAGCTCCTGCGCAGCCTTCA 3'
Cbs5
1174-1194
5' GTTCACCTTTGCCCGCATGCT 3'
Cbs6
1894-1914"
5' TGTTTAGGGCTCAGGAAAGCG 3'
PAH
CBS
Sequences of the oligonucleotides used as primers for amplification of PAH & CBS cDNAs and their relative location on the published sequence (11). Asterisk (*) represents the complementary primers of the coding sequence.
for rapid analysis of mutations in PAH and CBS. There have been several reports describing heterogeneous phenotypes of PKU and HCU. Screening of mutations in individuals with classic and less severe PKU and HCU may reveal clues about structure fimction relationship of these two important enzymes. We were able to amplify specific cDNAs corresponding to PAH and CBS mRNAs from RNA extracted from HepG2 cells and lymphocytes obtained from peripheral blood.
PCR
amplified products of expected size were seen after 1st round PCR on HepG2 cDNA but not with
646
Voi. 45, No. 4, 1998
BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL
lymphocyte cDNA (Fig. 1 & 2). This could be due to abundant expression of the PAH and CBS genes in the HepG2 cells. There were no visible products in the negative control (with out Reverse Transcriptase). Successful amplifications for both PAH and CBS resulted only after nested PCR, using 1st round PCR products of lymphocyte cDNA as a template, with the primer combinations of Pahl and Pah4; Cbsl and Cbs6 respectively. Occasionally faint bands other than expected size were observed in amplifications using lymphocyte cDNA as template. This may be due to non specific annealing of the primers. Table II describes the various primer combinations used for nested PCR ofPAH & CBS amplifications.
The results shown in figure
1A and 2A clearly demonstrate the successful amplification of both PAH and CBS products with individual primer sets. To confirm the authenticity of the amplifications, each of the individual products were digested using a unique restriction endonuclease cleavage site located in the respective sequences (Fig. 1B & 2B). The specificity was further confirmed by southern blot hybridization using the entire cDNAs of PAH and CBS obtained from HepG2 cells as probe
A
1'2
3 4 5 67
8
B
1 2 3
4
5
946 bp
' 817 bp _2 946 bp
563 bp
-" 817 bp --563bp
Figure 1: A. PCR amplifications using primers specific for PAH gene. Lanes 1 and 4 lymphocyte eDNA as template, lanes 2 and 5 1st round PCR products of Hep G2 eDNA as template, lanes 3 and 6 negative control (.RT) lane 7 , 1 st round PCR product with Pahl & Pah4 primers using lymphocyte eDNA as template and lane 8 molecular weight marker. Lanes 1, and 2 and 3 are products of Pah3/Pah4 and 4,5 and 6 are with Pahl/Pah2. B. Restriction digestion pattern of the products. Lanes 1 and 3 products from lymphocyte eDNA and 2 and 4 are from Hep G2 eDNA, 1and 2 are products of Pah3/Pah4 digested with Hind III, 3 and 4 are products of Pahl/Pah2 digested with Xba I, lane 5 molecular weight marker. 647
Vol. 45, No. 4, 1998
A
BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL
1 2 3 4 5 6 7 891011
S
1 2 3 4 5 6 7
946 bp 817bp
...,,946 bp "-"817 bp
563bp
- 563 bp
Figure 2: A. PCR amplifications using primers specific for CBS gene. PCR amplified products, lanes 1,4 and 7 lymphocyte eDNA as template, lanes 2,5 and 8 1st PCR products of Hep G2 eDNA as template, lanes 3,6 and 9 are negative control (-RT). Lanes 1,2 and 3 using primers Cbs5/Cbs6, 4,5 and 6 using primers Cbs3/Cbs4 and 7,8 and 9 using primers Cbsl/Cbs2. Lane 10 1st round PCR product of Cbsl & Cbs6 primers using lymphocyte cDNA as template. Lane 11 molecular weight marker. B. Restriction digestion pattern of the products. Lanes 1,3,5 lymphocyte products and 2,4 and 6 Hep G2 products. Lanes 1 and 2, digestion of Cbs5/Cbs6 products with Apa L I, lanes 3 and 4, digestions of Cbs3/Cbs4 products with Sac I, lanes 5 and 6 digestions of Cbsl/Cbs2 products with Sma I. Lane 7 molecular weight marker.
(data not shown). The yields were typically 1 - 2 ~tg from 35 rounds of nested PCR, sufficient for several sequencing reactions using the automated sequencer. The primers we have designed yielded products with reasonable size to obtain complete sequence from one reaction. Sequencing of the individual PCR products using respective primers was performed in an automated sequencer and the sequence generated was greater than 99% identical with the published sequence. There were some errors, which were corrected by analyzing the peaks manually and also the sequence of the opposite strand. There was ambiguity in some places, which may be a problem in heterozygote detection. However, this problem could be overcome by using fluorescein labeled primers for sequencing reactions where peak heights would be more reliable. We are currently synthesizing primers with fluorescein tags for this purpose. By using this technique, it is possible to screen mutations in the entire coding region and splice junctions in PAH & CBS.
Whether this technique can be used for prenatal diagnosis
648
Vol. 45, No. 4, 1998
BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL
Table - 2 Primer Combinations
Size of the PCR Products
Enzyme cleavage Sites
Size of the digested product (bp)
PAIl Pahl/Pah4
1451 bp
Pahl/Pah2
920 bp
XbaI
655,255
Pah3/Pah4
710 bp
HindlII
405,305
CBS CBS 1/CBS6
1820 bp
CBS 1/CBS2
662 bp
SmaI
497,165
CBS3/CBS4
620 bp
SacI
572,48
CBS5/CBS6
740 bp
ApaLI
541,199
using chorionic villus biopsy or cord blood remains to be determined. Our technique is less time consuming and cost effective than the ones currently used for mutation analysis of PAH and CBS which include exon amplifications and sequencing (16,17), RT-PCR on the RNAs isolated either from EBV transformed lymphocytes (9,10,18) or cultured fibroblasts (9,17).
In
conclusion we have described a rapid, reliable and simple technique to study mutations and cartier detection of PAH and CBS genes, using lymphocytes from whole blood. ACKNOWLEDGMENTS We are grateful to Professors M.R.S. Rao and S.K. Brahmachari for allowing us to use DBT central facilities for automated DNA sequencing and synthesis of oligonucleotides. K.S.D is recipient of Research Associateship from Council of Scientific and Industrial Research, New Delhi. We thank the Director, Indian Institute of Science, Bangalore 560 012 for financial support.
REFERENCES 1)
Chelly, J:, Concordet, J-P., Kaplan, J-C., and Khan, A. (1989) Proc Natl Acad Sci (USA). 86,2617-2621.
2)
Sarkar, G., and Sommer, S.S. (1989) Science. 244,331-334. 649
Vol. 45, No. 4, 1998
BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL
3)
Berg, L.P., Wieland, K., Miller, D.S., Schlosser, M., Wagner, M., Kakkar, V.V., Reiss, J., and Cooper, D.N. (1990) Hum. Genet. 85,655-658.
4)
Roberts, R.G., Barby, T.F.M., Manners, E., Bobrow, M., and Bentley, D.R. (1991) Am. J. Hum Genet. 49, 298-310.
5)
Roberts, R.G., Gardner, R.J., and Bobrow, M. (1994) Hum. Mutat. 4,1-11.
6)
Chalkley, G., and Harris, A. (1991) J. Med. Genet. 28,777-780.
7)
Fonknechten ,N., Chelly, J., Lepereq, J., Kahn, A., Kaplan, J.C., and Kitzis. (1992) Hum.Genet. 88,508-512.
8)
Romey, M.C., Tuffery, S., Desgeorges, M., Bienvenu, T., Demaille, J., and Claustres, M.(1996) Hum. Genet. 98,328-332.
9)
Ramus, S.J., Forrest, S.M., and Cotton, R.G.H. (1992) Hum. Mutat. 1,154-158.
10)
Tsai, M.Y., Bignell, M., Schwichtenberg, K., and Hanson, N.Q. (1996) Am. J. Hum. Genet. 59, 1262-1267.
11)
Kwok, S.C.M., Ledley, F.D., DiLella, A.G., Robson, K.J.H., and Woo, S.L.C. (1985) Biochemistry. 24, 556-561.
12)
DiLella, A., Kwok, S., Ledley, F., Marvit, J., and Woo, S.L.C. (1986) Biochemistry. 25, 743 -749.
13)
Kraus, J.P., Le, K., Swaroop, M., Ohura, T., Tahara, T., Rosenberg, L.E., Roper, M.D., and Kozich, V. (1993) Hum. Mol. Genet. 2(10), 1633-1638.
14)
Kery, V., Bukovska, G., and Kraus, J.P. (1994) J. Biol. Chem. 269, 25283-25288.
15)
Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159.
16)
Eisensmith, R.C., and Woo, S.L.C. (1995) in Advances in Genetics. (Hall JC and Dunlop JC. Eds.), Vol. 32 pp. 199-271, Academic Press, Inc., New York, USA.
17)
Sebastio, G., Sperandeo, M.P., Panico, M., de Franchis, R., Kraus, J.P., and Andria, G. (1995) Am. J. Hum. Genet. 56, 1324-1333.
18)
Shih, V.E., Fringer, J.M., Mandell, R., Kraus, J.P., Berry, G.T., Heidenreich, R.A., Ko,M.S., Levy, H.L., and Ramesh, V. (1995) Am. J. Hum. Genet. 57, 34-39
650
