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Allele Separation Facilitates Interpretation of Potential Splicing Alterations and Genomic Rearrangements¹

Human Cancer Genetics Program, Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210 [H. N., J. L., H. H., A. d. l. C.]; and Howard Hughes Medical Institute, The Oncology Center, The Johns Hopkins University, Baltimore, Maryland 21231 [H. Y., K. W. K., B. V.]

	ABSTRACT

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Mutations that alter normal splice patterns and genomic rearrangements are common causes of hereditary diseases including hereditary nonpolyposis colorectal cancer. However, abnormal transcripts can be difficult to detect and interpret because splicing patterns are often heterogeneous even in normal cells. Standard techniques including sequencing and Southern hybridization fail to detect some genomic rearrangements. We show here that separation of alleles in somatic cell hybrids, through "conversion" technology, considerably facilitates the interpretation of abnormal splicing patterns and the detection of genomic rearrangements. We detected novel mutations in MLH1 in each of four hereditary nonpolyposis colorectal cancer patients. The genomic mutations were CAG>CAA predicting Q346Q; GAG>AAG predicting E102K; a>g at nucleotide 1559–2 at intron 13, and a tandem duplication involving exons 7–12. By separating the two alleles, we showed that one allele produced only abnormal transcript or no transcript whereas the other allele produced only normal transcript. These results allowed pathogenicity to be unambiguously assigned to the mutations and increased the sensitivity of genomic testing.

	Introduction

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Mutation detection is key to the diagnosis of inherited disorders. In some diseases, searching for one or a small number of recurrent or widespread mutations is sufficient for most diagnostic purposes. For example, in sickle cell anemia most patients have a missense founder mutation of the ß-globin gene (1) , and in achondroplasia almost all patients have a recurrent missense mutation of the fibroblast growth factor receptor-3 gene (2) . By contrast, in many diseases the phenotype results from a variable number of different mutations. Moreover, some diseases are genetically heterogeneous in that mutations in more than one gene can cause the phenotype. For instance, in HNPCC³ , germ-line mutations of at least four mismatch repair genes have been implicated (MLH1, MSH2, MSH6, PMS2; Refs. 3 and 4 ). The total number of different mutations in these genes is presently greater than 300,⁴ and the list is rapidly growing (3) .

In 20–50% of putative HNPCC families and patients, no mutation is detectable by standard methods (5, 6, 7) . Many methods have been devised to improve mutation detection (8 , 9) ; however, one class of changes remains particularly difficult to detect and interpret (i.e., rearrangements of the transcripts, many of which lead to the decrease or disappearance of the transcript through NMD; Ref. 10 ). These mutations mainly consist of sequence changes causing splicing errors or consist of large genomic rearrangements. The clinical significance of abnormal transcripts is particularly difficult to interpret in genes where alternatively spliced isoforms of the transcript occur naturally and frequently. One such gene is MLH1 (11 , 12) , which shows several alternative splicing patterns. In this study, we show that the separation of alleles in somatic cell hybrids (here referred to as conversion; Ref. 13 ) considerably facilitates the detection and interpretation of abnormal transcripts.

	Materials and Methods

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Patients and Samples.
Four unrelated patients belonging to HNPCC families fulfilling the Amsterdam criteria (4) , each with a microsatellite unstable colorectal carcinoma, were studied. Three of the patients were previously mentioned in the initial report on the conversion technology (13) as: patient 1 (ML), patient 2 (CG), and patient 3 (GS). Patient 4 has not been published previously. For each patient, an EBV-transformed lymphoblastoid cell culture was available as a source of DNA and RNA. In patients 1–3 lymphoblastoid cells were used to produce the fusion clones converted to haploidy, whereas in patient 4 lymphocytes from a fresh blood sample were used for this purpose.

Diploid-to-Haploid Conversion.
Haploid-converted clones of patients 1–3 were created by using the conversion technology of Yan et al. (13) . Haploid-converted clones from the fourth patient were created by GMP Genetics, Inc. In brief, human fresh lymphocytes or established lymphoblastoid cells were electrofused with a specifically designed mouse cell line (E2). Unfused mouse parental cells were negatively selected by sodium hypoxanthine, aminopterin, and thymidine (HAT; Life Technologies, Inc.), and unfused human lymphocytes were negatively selected by Geneticin (Life Technologies, Inc.). Hybrid cells were maintained in DMEM (Life Technologies, Inc.) including 10% FBS, 0.5 mg/ml Geneticin, 1x HAT, and penicillin-streptomycin.

RT-PCR Analysis.
RNA was extracted from original lymphoblastoids and converted cells by Trizol according to the manufacturer (Life Technologies, Inc.). Two micrograms of total RNA were treated with Superscript II (Life Technologies, Inc.) to produce cDNA, using random hexamers according to the manufacturer. We designed four sets of primers to amplify the entire coding sequence (a) in a single amplicon and (b) as three overlapping amplicons. These were designed so as not to amplify mouse MLH1. The primers for the amplification of the entire coding sequence were 5'-gcgccaaaatgtcgttcgtg-3'(Ex1F) and 5'-cccacagtgcataaataacc-3'(Ex19R). The primers for the first part of MLH1 were Ex1F and 5'-ctgagtagtttgcattggatatgtaa-3'(Ex9R), for the second part 5'-gagacagtagctgatgttaggacacta-3'(Ex8F) and 5'-cagtcatttcctttcgggaat-3'(Ex13R), and for the third part 5'-agaggacctacttccagcaacc-3'(Ex12F) and Ex19R. Other primers used for amplification are available on request. Long-range RT-PCR was performed by using Expand Long Template PCR system (Roche), and PCR was carried out at 94°C (10 s), 60°C (30 s), and 68°C (2 min) for 10 cycles, and at 94°C (10 s), 60°C (30 s), and 68°C (2 min + cycle elongation for more yields of 20 s for each cycle) for 25 cycles. The PCR products were run on a 1.5% agarose gel to analyze their size differences.

DNA Sequencing.
The RT-PCR products were isolated using QIAquick PCR product purification kit (Qiagen). The purified RT-PCR products were sequenced directly by the primer used for PCR, or cloned into TA vector pCR2.1 (Invitrogen). DNA from plasmid clones was extracted by QIAprep Spin Miniprep kit (Qiagen) and sequenced using the ABI sequencing system (Perkin-Elmer Applied Biosystems). Amplification of the exons and exon-intron junctions of MLH1 and sequencing was described previously (14) .

	Results and Discussion

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Genomic Sequencing.
Exon-by-exon genomic sequencing including the promoter regions of MLH1 and MSH2 revealed no changes in lymphoblast DNA from cases 2 and 3. In case 1, there was an a to g change at the conservative splice acceptor site of exon 14 of MLH1 (ag at nucleotide 1559-2). In case 4, there was a G to A change at codon 102 predicting a glutamic acid to lysine, E102K, amino acid substitution of unknown significance.

RT-PCR of Diploid Cell Transcripts.
Using RNA from lymphoblastoid cells, in all four cases a normal-sized transcript was seen by RT-PCR; in addition, in cases 1–3, a very faint aberrant band was also seen (Fig. 1A) . In case 4, only a normal-sized transcript was seen in the diploid cells (Fig. 1A) .

View larger version (49K): [in this window] [in a new window]   Fig. 1. A, RT-PCR analysis of the original lymphoblastoid cells (Ly), the hybrid cells with wild-type allele (W), and the hybrid cells with mutant allele (M). The four lanes labeled n.c. (normal control) are from lymphocytes from healthy individuals, and E2 is the recipient mouse cells used in the conversion. SM, size marker. In case 1, the M lanes display very weak expression of a 739-bp abnormal transcript and no normal transcript. In case 2, the abnormal transcript is larger (267 bp) than the normal transcript (208 bp). In case 3, the M allele is larger (2.5 kb) than the W transcript (1.6 kb). In case 4, W and M cells express almost same-sized transcripts of equal quantity. B, sequences of the abnormal transcripts. In case 1, exons 14 and 15 are missing. In case 2, there is a 59-bp insertion between exons 11 and 12. The sequence is derived from intron 11 (intron inclusion). In case 3, there is a tandem duplication of exons 7–12. In case 4, there is a 5-bp deletion (GTAAG) at the junction of exon 3 and intron 3. C, sequences of the wild-type and mutant alleles as described in B. In case 1, the abnormal g in the mutant allele is boxed. In case 2, the boxed mutant A is the last nucleotide of exon 11. In case 3, 15-kb regions containing exons 7–12 are duplicated, and it occurred as a result of homologous recombination between introns 6 and 12, in which 280 bp (arrowhead) of Alu sequence occurs. In case 4, the mutant A (boxed) is the third nucleotide from the 3' end of exon 3. The new splice site is shown with a dashed line.

In case 1 (Fig. 1) , RT-PCR demonstrated only normal transcript in the W clones and only a smaller than normal transcript in the M clones; this transcript was of extremely weak abundance, apparently as a consequence of NMD. Sequencing revealed that this transcript lacked exons 14 and 15. The ag change at nucleotide 1559-2 seen previously in heterozygous form in lymphoblastoid cell DNA occurred in the M clones but not in the W clones. Thus, this change apparently caused the splicing error.

In case 2 (Fig. 1) , RT-PCR showed a normal product in W clones but a larger product in M clones. By sequencing, a 59-bp insertion emanating from the 5' end of intron 11 was seen in this transcript. Sequencing of the M clones revealed a GA change in the last nucleotide of exon 11 that had not been noticed in the sequencing of lymphoblastoid cell DNA. This change obviously abrogates the splicing that instead occurs at the next downstream splicing-donor consensus gt in intron 11, resulting in the addition of 59 bp to the transcript. As can be seen (Fig. 1A) in lymphoblastoid cells, the abnormal transcript is present in greatly reduced quantity compared with the wild-type transcript, hence it is subject to NMD.

In case 3 (Fig. 1) , RT-PCR showed normal transcript in W clones but only an abnormally large transcript in M clones. By sequencing, this turned out to represent a tandem duplication of exons 7–12. By genomic sequencing of the M clones, a 15-kb tandem duplication comprising exons 7–12 was identified. It had arisen through homologous recombination between introns 6 and 12 in Alu regions showing 88% sequence identity. For unknown reasons, Southern blot analysis by using a cDNA probe of MLH1 against HindIII- and NsiI-digested genomic DNA did not detect this genomic rearrangement of MLH1 (data not shown).

In case 4 (Fig. 1) , RT-PCR showed a normally sized product of MLH1 in both W clones and M clones. By sequencing of the transcript from the M clones, a 5-bp deletion in the 3' end of exon 3 was present as well as the minor population of the normally sized transcript with one nucleotide change. Sequencing of genomic DNA from the M clones revealed the missense mutation at codon 102 (GAGAAG) that had been noticed in the sequencing of lymphocyte DNA. This nucleotide change abrogates the splicing pattern of exon 3, which instead occurs 5 bp upstream within exon 3, resulting in a 5-bp deletion in the transcript. This change is obviously pathogenic.

Table 1 summarizes the findings. Without conversion, a mutation was signaled in two of the cases by genomic sequencing and in three of the cases by RT-PCR. However, in all four cases it was the study of the haploid-converted clones that made it possible to delineate and assess the potential disease-causing role of the mutations. In particular, the presence of one or several minor species of transcript, as seen in cases 1–3, is hard to assess in the diploid context. This is the case even when the sequence of the minor species can be determined. In contrast, by demonstrating that the abnormal transcript emanates from one of the alleles as shown in all four cases here, and that it is of greatly diminished quantity, as shown in 2 cases here, a decisive, deleterious role is likely.

	ACKNOWLEDGMENTS

 
We thank Tamara Hemingway and Getachew Boru for assistance with cell culture and DNA preparation and Barbara Fersch for preparation of this manuscript.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by Grants CA67941, CA16058, CA43460, and CA62924 from the National Institutes of Health. H.N. is a postdoctoral fellow from Japan Society for Promoting of Science and is supported by Sumitomo Life Social Welfare Service’s Foundation, The Osaka Medical Research Foundation for Incurable Disease, and Yamanouchi Foundation for Research on Metabolic Disorders. Under agreements between The Johns Hopkins University and GMP Genetics, Inc., K. W. K. and B. V. are entitled to a share of the royalties received by the University on sales of products related to the use of Conversion technologies. K. W. K. and B. V. are consultants to GMP Genetics, Inc. The University, K. W. K., and B. V. also own stock in GMP Genetics, Inc., which is subject to certain restrictions under University policy. The terms of these arrangements are being managed by the University in accordance with its conflict of interest policies.

² To whom requests for reprints should be addressed, at Human Cancer Genetics Program, Comprehensive Cancer Center, The Ohio State University, Medical Research Facility, Room 646, 420 West 12th Avenue, Columbus, OH 43210. Phone: (614) 688-4781; Fax: (614) 688-4772; E-mail: delachapelle-1{at}medctr.osu.edu

³ The abbreviations used are: HNPCC, hereditary nonpolyposis colorectal cancer; NMD, nonsense-mediated decay; M, mutated; W, wild type.

⁴ Internet address: www.nfdht.nl/.

Received 4/18/02. Accepted 6/24/02.

	REFERENCES

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