Letters to the Editor References Martin NG, Olsen ME, Theile H, El Beani JL, Handelsman D, Bhatnagar AS (1984) Pituitary-ovarian function in mothers who have had two sets of dizygotic twins. Fertil Steril 41:878-880 Reed TE (1990) Age at menarche as a fitness trait: further considerations. Am J Hum Genet 48:420-421 Treloar SA, Martin NG (1990) Age at menarche as a fitness trait: nonadditive genetic variance detected in a large twin sample. Am J Hum Genet 47:137-148
423 B
A
b
a ..j627bp --*-474bp .p
c
d
_-627bp-m-
*.-474bp 'N4l428bt
_-153bp-i
i 1991 by The American Society of Human Genetics. All rights reserved. 0002-9297/91 /4802-0028$02.00
Us Am. J. Hum. Genet. 48:423-424, 1991
Heterozygotes and Homozygotes: Discrimination by Chemical Cleavage of Mismatch To the Editor:
The chemical reactivity, in a heteroduplex formed from wild-type and mutant DNA, of mismatched T and C bases with, respectively, osmium tetroxide and hydroxylamine is a useful tool for scanning mutations in the genome (Cotton et al. 1988). However, the definition of the homozygous or heterozygous state of the mutation is labor intensive and time consuming, being based on sequencing techniques. We report here a simple and efficient approach for identifying the status of homozygosity or heterozygosity for a specific mutation by using the chemical cleavage of mismatch (CCM) method. Genomic DNA from two I-thalassemic patients and from a normal control was amplified using appropriate primers to obtain a 627-bp fragment encompassing exons 1 and 2 of the 3-globin gene. By allele-specific oligonucleotide analysis, patient A was identified as homozygous for a nonsense C-*T mutation at codon 39, and patient B was identified as a compound heterozygote for the same mutation and for a G--A substitution at position 110 of intron 1 (IVS 1-110) (Kazazian and Bohem 1988). Both changes are identifiable by hydroxylamine reactions; in the case of codon 39 a C-A mismatch is formed in the heteroduplex by the sense strand of the probe, whereas in the case of IVS 1-110 a C-A mismatch is originated by the antisense DNA strand. Internally labeled probes were prepared from DNA of both the
F igu re I A, CCM analysis in patient homozygous for C-~T transition at codon 39 of 13-globin gene. The patient/control reaction (track a) shows the expected mismatch. As the original PCR product had been cleaved at the position of the mismatch, two cleavage products are found. The patient/patient reaction (track b) does not show any reactivity, as both alleles carry the same mutated sequence. B, CCM analysis in patient compound heterozygous for codon 39 mutation and for intronic G-'A transition (IVS 1-110). This patient carries a normal IVS I- 1 10 allele and a mutant codon 39 allele on one chromosome, and on the other carries a mutant IVS I-itO1 allele and a normal codon 3 9 allele. Each mutation results in two cleavage products, so four bands are found in the autoradiograph when a probe with a normal sequence is used (track c). The presence of the normal sequences in the labeled probe obtained from the patient DNA results in the patient/patient reaction (track d) having a pattern identical to that of the control/patient (track c) and identifies the condition of heterozygote (as in the case of the control/patient reaction the two C-A mismatches are cleaved on opposite DNA strands).
patients and the normal control by adding 20 pmol of 32P dCTP at the 25th cycle of PCR and performing 10 more cycles. The labeled PCR products were subsequently purified by means of PAGE. Heteroduplexes were formed by annealing the labeled and the unlabeled DNA fragments according to a method described elsewhere (Cotton et al. 1988) and were reacted for 1-3 h with hydroxylamine. The unlabeled PCR fragment from each patient was first hybridized to a probe from a normal subject to detect the mismatches. Subsequent hybridization to a probe prepared from the patient himself allowed heterozygosity or homozygosity to be determined. The results are shown in figure 1. Hybridizing probe from patient A to DNA from patient A did not show any reactivity after CCM analysis (fig. 1, track b). This confirms that only one allele type is present and that the patient is
Letters to the Editor
424
homozygous for the codon 39 mutation. Hybridizing probe from patient B to DNA from the patient himself (fig. 1, track d) gave a mismatch pattern identical to the pattern observed when the normal probe was used (fig. 1, track c). This implies that the patient carries both normal and mutant sequences at this locus, i.e., is a heterozygote. We used two 1-thalassemia mutations very common in Mediterranean populations and both leading to C-A mismatches, as a model, but the test may be performed also for all the other types of mismatches cleaved either by hydroxylamine or by osmium tetroxide. This test may extend the use of the CCM method to prenatal diagnosis of recessive hereditary conditions, whenever the parents are carriers of the same genetic lesion. If the parents are carriers of different mutations, the discrimination between homozygosity and heterozygosity is directly feasible by use of the normal probe (Dianzani et al. 1990). IRMA DIANZANI, *§ SUSAN M. FORREST,§ CLARA CAMASCHELLA,t $ ENRICO GOTTARDI,t AND RICHARD G. H. COTTON§ *Istituto di Clinica Pediatrica, and tDipartimento Scienze Biomediche e Oncologia Umana, and tCNR Centro di Immunogenetica e Istocompatibilita, Turin; and §The Murdoch Institute, Royal Children's Hospital, Parkville, Victoria, Australia References Cotton RGH, Rodrigues NR, Campbell RD (1988) Reactivity of cytosine and thymine in single-base-pair mismatches with hydroxylamine and osmium tetroxide and its application to the study of mutations. Proc Natl Acad Sci USA 85:4397-4401 Dianzani I, Camaschella C, Gottardi E, Forrest SM, Ramus S, Cotton RGH (1990) Simultaneous screening of the Mediterranean 13 thalassemia mutations by chemical cleavage of mismatch. Am J Hum Genet 47 [Suppl]: A214 Kazazian HH Jr, Bohem CD (1988) Molecular basis and prenatal diagnosis of 13 thalassemia. Blood 72:1107-1116 © 1991 by The American Society of Human Genetics. All rights reserved. 0002-9297/91 /4802-0029$02.00
Am. J. Hum. Genet. 48:424-425, 1991
Uniparental Disomy and Gene Localization To the Editor:
Uniparental heterodisomy, mentioned as a possible of genetic imprinting in the recent review by Hall (1990), has recently been demonstrated to be one possible cause of the Prader-Willi syndrome (Nicholls et al. 1989) and of male-to-male transmission of hemophilie A (Vidaud et al. 1989). Because of recombination prior to nondisjunction, Prader-Willi patients might also show uniparental isodisomy for one segment and heterodisomy for another segment of chromosome 15 (W. Robinson, personal communication), and this holds true, of course, for any uniparental disomy. Uniparental isodisomy is one possible interpretation (incest being another) for the discovery of homozygosity for an inv(4) chromosome in a child born to a heterozygous mother (Carpenter et al. 1982). Chromosome examination in the child was performed because of mental retardation. Uniparental disomy is also a possible explanation (mosaicism being another) for two instances of transmission of a Robertsonian 22/22 translocation from a mother to her daughter (Kirkels et al. 1980; Palmer et al. 1980), and for the nontransmission of a Robertsonian 15/ 15 translocation from a father to his son (paternity most likely; Lipson and Breg 1978). This latter case is of interest, as the child did not present the Prader-Willi phenotype despite possibly having two maternal chromosomes 15. In addition, maternal isodisomy was found in two patients with cystic fibrosis (CF) on molecular investigation of the family: all informative molecular probes on chromosome 7 were found to be of maternal origin (Spence et al. 1988; Voss et al. 1989). These two children, in addition to suffering from CF, were of very short stature, a feature that is not usually associated with this disease when treated properly. Although the incidence of uniparental hetero- and isodisomy for any chromosome is not known, the above-mentioned examples indicate that it might be frequent enough to justify examinations in the following, particularly interesting situations: 1. Patients with rare recessive disorders who, in addition, have one or several features uncommon to their disorder, particularly growth retardation and/ or mental deficiency. When the gene localization of the disorder is not known, one would have to investigate at least one highly polymorphic marker for each of the 22 autosomes in child and parents cause
423 B
A
b
a ..j627bp --*-474bp .p
c
d
_-627bp-m-
*.-474bp 'N4l428bt
_-153bp-i
i 1991 by The American Society of Human Genetics. All rights reserved. 0002-9297/91 /4802-0028$02.00
Us Am. J. Hum. Genet. 48:423-424, 1991
Heterozygotes and Homozygotes: Discrimination by Chemical Cleavage of Mismatch To the Editor:
The chemical reactivity, in a heteroduplex formed from wild-type and mutant DNA, of mismatched T and C bases with, respectively, osmium tetroxide and hydroxylamine is a useful tool for scanning mutations in the genome (Cotton et al. 1988). However, the definition of the homozygous or heterozygous state of the mutation is labor intensive and time consuming, being based on sequencing techniques. We report here a simple and efficient approach for identifying the status of homozygosity or heterozygosity for a specific mutation by using the chemical cleavage of mismatch (CCM) method. Genomic DNA from two I-thalassemic patients and from a normal control was amplified using appropriate primers to obtain a 627-bp fragment encompassing exons 1 and 2 of the 3-globin gene. By allele-specific oligonucleotide analysis, patient A was identified as homozygous for a nonsense C-*T mutation at codon 39, and patient B was identified as a compound heterozygote for the same mutation and for a G--A substitution at position 110 of intron 1 (IVS 1-110) (Kazazian and Bohem 1988). Both changes are identifiable by hydroxylamine reactions; in the case of codon 39 a C-A mismatch is formed in the heteroduplex by the sense strand of the probe, whereas in the case of IVS 1-110 a C-A mismatch is originated by the antisense DNA strand. Internally labeled probes were prepared from DNA of both the
F igu re I A, CCM analysis in patient homozygous for C-~T transition at codon 39 of 13-globin gene. The patient/control reaction (track a) shows the expected mismatch. As the original PCR product had been cleaved at the position of the mismatch, two cleavage products are found. The patient/patient reaction (track b) does not show any reactivity, as both alleles carry the same mutated sequence. B, CCM analysis in patient compound heterozygous for codon 39 mutation and for intronic G-'A transition (IVS 1-110). This patient carries a normal IVS I- 1 10 allele and a mutant codon 39 allele on one chromosome, and on the other carries a mutant IVS I-itO1 allele and a normal codon 3 9 allele. Each mutation results in two cleavage products, so four bands are found in the autoradiograph when a probe with a normal sequence is used (track c). The presence of the normal sequences in the labeled probe obtained from the patient DNA results in the patient/patient reaction (track d) having a pattern identical to that of the control/patient (track c) and identifies the condition of heterozygote (as in the case of the control/patient reaction the two C-A mismatches are cleaved on opposite DNA strands).
patients and the normal control by adding 20 pmol of 32P dCTP at the 25th cycle of PCR and performing 10 more cycles. The labeled PCR products were subsequently purified by means of PAGE. Heteroduplexes were formed by annealing the labeled and the unlabeled DNA fragments according to a method described elsewhere (Cotton et al. 1988) and were reacted for 1-3 h with hydroxylamine. The unlabeled PCR fragment from each patient was first hybridized to a probe from a normal subject to detect the mismatches. Subsequent hybridization to a probe prepared from the patient himself allowed heterozygosity or homozygosity to be determined. The results are shown in figure 1. Hybridizing probe from patient A to DNA from patient A did not show any reactivity after CCM analysis (fig. 1, track b). This confirms that only one allele type is present and that the patient is
Letters to the Editor
424
homozygous for the codon 39 mutation. Hybridizing probe from patient B to DNA from the patient himself (fig. 1, track d) gave a mismatch pattern identical to the pattern observed when the normal probe was used (fig. 1, track c). This implies that the patient carries both normal and mutant sequences at this locus, i.e., is a heterozygote. We used two 1-thalassemia mutations very common in Mediterranean populations and both leading to C-A mismatches, as a model, but the test may be performed also for all the other types of mismatches cleaved either by hydroxylamine or by osmium tetroxide. This test may extend the use of the CCM method to prenatal diagnosis of recessive hereditary conditions, whenever the parents are carriers of the same genetic lesion. If the parents are carriers of different mutations, the discrimination between homozygosity and heterozygosity is directly feasible by use of the normal probe (Dianzani et al. 1990). IRMA DIANZANI, *§ SUSAN M. FORREST,§ CLARA CAMASCHELLA,t $ ENRICO GOTTARDI,t AND RICHARD G. H. COTTON§ *Istituto di Clinica Pediatrica, and tDipartimento Scienze Biomediche e Oncologia Umana, and tCNR Centro di Immunogenetica e Istocompatibilita, Turin; and §The Murdoch Institute, Royal Children's Hospital, Parkville, Victoria, Australia References Cotton RGH, Rodrigues NR, Campbell RD (1988) Reactivity of cytosine and thymine in single-base-pair mismatches with hydroxylamine and osmium tetroxide and its application to the study of mutations. Proc Natl Acad Sci USA 85:4397-4401 Dianzani I, Camaschella C, Gottardi E, Forrest SM, Ramus S, Cotton RGH (1990) Simultaneous screening of the Mediterranean 13 thalassemia mutations by chemical cleavage of mismatch. Am J Hum Genet 47 [Suppl]: A214 Kazazian HH Jr, Bohem CD (1988) Molecular basis and prenatal diagnosis of 13 thalassemia. Blood 72:1107-1116 © 1991 by The American Society of Human Genetics. All rights reserved. 0002-9297/91 /4802-0029$02.00
Am. J. Hum. Genet. 48:424-425, 1991
Uniparental Disomy and Gene Localization To the Editor:
Uniparental heterodisomy, mentioned as a possible of genetic imprinting in the recent review by Hall (1990), has recently been demonstrated to be one possible cause of the Prader-Willi syndrome (Nicholls et al. 1989) and of male-to-male transmission of hemophilie A (Vidaud et al. 1989). Because of recombination prior to nondisjunction, Prader-Willi patients might also show uniparental isodisomy for one segment and heterodisomy for another segment of chromosome 15 (W. Robinson, personal communication), and this holds true, of course, for any uniparental disomy. Uniparental isodisomy is one possible interpretation (incest being another) for the discovery of homozygosity for an inv(4) chromosome in a child born to a heterozygous mother (Carpenter et al. 1982). Chromosome examination in the child was performed because of mental retardation. Uniparental disomy is also a possible explanation (mosaicism being another) for two instances of transmission of a Robertsonian 22/22 translocation from a mother to her daughter (Kirkels et al. 1980; Palmer et al. 1980), and for the nontransmission of a Robertsonian 15/ 15 translocation from a father to his son (paternity most likely; Lipson and Breg 1978). This latter case is of interest, as the child did not present the Prader-Willi phenotype despite possibly having two maternal chromosomes 15. In addition, maternal isodisomy was found in two patients with cystic fibrosis (CF) on molecular investigation of the family: all informative molecular probes on chromosome 7 were found to be of maternal origin (Spence et al. 1988; Voss et al. 1989). These two children, in addition to suffering from CF, were of very short stature, a feature that is not usually associated with this disease when treated properly. Although the incidence of uniparental hetero- and isodisomy for any chromosome is not known, the above-mentioned examples indicate that it might be frequent enough to justify examinations in the following, particularly interesting situations: 1. Patients with rare recessive disorders who, in addition, have one or several features uncommon to their disorder, particularly growth retardation and/ or mental deficiency. When the gene localization of the disorder is not known, one would have to investigate at least one highly polymorphic marker for each of the 22 autosomes in child and parents cause
