American Journal of Medical G e n e t i c s 44356-360 (1992)
Clinical Phenotype and Molecular Analysis of a Three-Generation Family With an Interstitial Deletion of the Short Arm of Chromosome 5 L. D. Keppen, S. M. Gollin, D. Edwards, J. Sawyer, W.Wilson, and J. Overhauser Department of Pediatrics, University of South Dakota School of Medicine, Sioux Falls (L.D.K.); Department of Human Genetics, University of Pittsburgh, Pittsburgh (S.M.G.) and Department of Biochemistry and Molecular Biology, Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia (W.W., J.O.), Pennsylvania; and Departments of Pediatrics (D.E.) and Pathology (J.S.), UAMS, Little Rock, Arkansas (J.S.) do not seem to significantly alter the phenotype other than demonstrating a trend toward a greater degree of microcephaly,growth failure, and cognitive impairment [Wilkins, 1981; Wilkins et al., 1982; Carlin, 198911. Deletions that do not include the 5p15.2 critical region can result in varied clinical manifestations. An individual and several offspring with an interstitial deletion extending from 5p14 to 5p15.1 [de1(5)(5qter->5 p14::5p15.2->5pter)l had only microcephaly and mild mental retardation [Walker et al., 1984; Overhauser et al., 1986bl. In addition, 6 individuals in a 3-generation family with an interstitial deletion of 5p14[de1(5) (5qter->5p14::5~15.1>pterll were completely asymp0 1992 Wiley-Liss, Inc. tomatic and are mentally normal [Overhauser et al., 1986a1. The observation that 5p deletions can give rise to a wide range of clinical phenotypes demonstrates the KEY WORDS: chromosome 5, deletion, menneed to understand more clearly the role of each chromotal retardation, cri-du-chat somal region in the appearance of clinical manifestasyndrome tions and would allow for more accurate prognosis after prenatal diagnosis or the discovery ofa 5p deletion after birth. INTRODUCTION Here we describe a 3-generation family with an interDeletions in the short arm of chromosome 5 (5p) usu- stitial deletion of 5p that includes 5p14 and a portion of ally are associated with the cri-du-chat syndrome, char- 5p13. Clinical findings and comparison with previously acterized by a high-pitched, monochromatic cry; crani- published interstitial deletions are discussed. ofacial abnormalities, which include microcephaly, apparent hypertelorism, and micrognathia; a slowed CLINICAL REPORT rate of growth; and psychomotor retardation [Niebuhr, The family pedigree is shown in Figure 1.The propo197813; Carlin, 19891. Although the size of the deleted situs (111-1) was a small-for-gestational age infant with segment varies from patient to patient, there appears to microcephaly born to a 35-year old moderately retarded be a critical segment at 5p15.2 that is deleted in all mother. Birth weight was 1,630 g at 34 weeks of gestapatients with the cri-du-chat syndrome [Niebuhr, tion. At age 4 years, he has moderate mental retarda1978a; Overhauser et al., 1986bl. Larger deletions, tion, severe microcephaly,esotropia and hyperopia, midwhich include the loss of 5p material other than 5~15.2, face hypoplasia, and hypertonia (Fig. 2). His moderately retarded mother (11-4) has microcephaly, simple ears, micrognathia, scoliosis,and arachnodactyly. She can communicate only minimally, canReceived for publication December 9, 1991; revision received not read or write, and has an I& of 50. The maternal April 14, 1992. grandmother (1-1) was mildly mentally retarded and Address reprint requests to Joan Overhauser, Ph.D., Department of Biochemistry and Molecular Biology,Jefferson Institute of had kyphoscoliosis. The grandmother, mother, and propositus each had an Molecular Medicine, Thomas Jefferson University, 233 South 10th interstitial deletion of 5p. High-resolution chromosome Street, Philadelphia, PA 19107. We report on a %generation family with an interstitial deletion of the short a r m of chromosome 5. Varied manifestations were found among the affected individuals including microcephaly, hypertonia, and micrognathia; mental retardation was common to all affected individuals. High resolution chromosome analysis was interpreted as del(5) (pter->p14.3::~13.3->qter).Molecular comparison of the deletion in this family with individuals with other 5p deletions suggests that the clinical findings are due specifically to the chromosomal material deleted from 5p13.
0 1992 Wiley-Liss, Inc.
Interstitial Deletion of 5p
357
Y’
I
111
Fig. 1. Family pedigree. Left half of the symbol filled in represents cytogenetic confirmation of 5p deletion. Top right quarter filled in represents mental retardation. Bottom right quarter filled in represents mosaic trisomy 8.
analysis was interpreted as del(5)(pter->p14.3::~13.3-> qter) in the grandmother, mother, and propositus (Fig. 3). The grandmother has had 5 pregnancies. The oldest daughter (11-1) is more severely retarded than the mother of the propositus. She is said to have a facial appearance similar to that of the mother of the propositus and is in foster care. The second pregnancy resulted in a normal daughter (11-2).The third pregnancy was a spontaneous abortion at 4-5 months of gestation. The mother of the propositus was the result of the fourth pregnancy. The fifth pregnancy was a normal male who is 31 years old. It was difficult to evaluate the grandmother’s intelligence since she had Alzheimer disease. It was initially thought that she most likely had a balanced translocation since she did not have microcephaly and had reared her children, but cytogenetic abnormalities were not identified by repeated high resolution chromosome analysis. It was not possible t o get permission for a skin biopsy to rule out mosaicism. Other relatives were not available for evaluation. The mother later had another son (111-2) who had mosaic trisomy 8 in addition to deletion 5p. This was
Fig. 2. The propositus.
confirmed by fibroblast cell culture. Analysis of 30 trypsin-Giemsa banded metaphase cells from PHA-stimulated peripheral blood leukocyte cultures showed 7 cells (23%) with a 46,XY,de1(5)chromosome pattern and 23 cells (77%)with a 47,XY 8,de1(5)chromosome pattern. Analysis of 20 trypsin-Giemsa banded metaphase cells from tissue fibroblast cell cultures showed 8 cells (40%) with a 47,XY, + 8,de1(5) chromosome pattern and 12 cells (60%) with a 46,XY,de1(5) chromosome pattern. There was no evidence of mosaicism in the propositus by tissue fibroblast or lymphocyte cell culture. The son with del(5p) and mosaic trisomy 8 had severe
+
8
5 ---I
I
A
B
C
D
Fig. 3. Representative pairs of chromosome 5 from all affected relatives showing the interstitial deletion, del(5Kp13.3p14.3).A: 1-1,B:II-4;C:III-1;D:III-2.Arrows indicate position of the deletion. Dotted lines indicate centromere position.
358
Keppen et al.
mental retardation, microcephaly, downward slanting palpebral fissures, hyperopia and esotropia, thin, incompletely infolded ear helices, and 5th digit clinodactyly. He had hypertonia and has had poor weight gain. Based on the clinical picture of individuals with this 5p deletion, most of his abnormalities and the greater severity of mental retardation are due to mosaic trisomy 8. The microcephaly and mental retardation appear to be the only clinical features due to the 5p deletion. The presence of the mosaic trisomy 8 is unlikely to be related to the 5p deletion. Both sons were placed in foster care at the time of nursery discharge because the mother was unable to care for them. MATERIALS AND METHODS An Epstein-Barr virus-transformed cell line was established on individual 11-4, which was used to make somatic cell hybrid lines that specifically retained the deleted chromosome 5. The procedures used to make these human-Chinese hamster cell hybrids have been described in detail elsewhere [Dana and Wasmuth, 1982; Overhauser et al., 1986bl. The resulting somatic cell hybrids (JH151 and JH162) were analyzed using GTG banding to confirm the specific retention of the deleted chromosome 5. Additional somatic cell hybrids were used for comparison studies to map the chromosome breakpoints in the present family relative to other previously analyzed 5p deletions. Hybrid HHW213 contains the entire short arm of chromosome 5 and a small portion of the long arm [Dana and Wasmuth, 19821. Somatic cell hybrids HHW339, JH141, and JH132 are derived from patients with cri-du-chat syndrome and have terminal deletions at 5~15.1,5p13, and 5p13, respectively. The precise mapping of these breakpoints was determined using cytogenetic techniques (GTG-banding)as well as physical mapping using random DNA fragments from the short arm of chromosome 5 [Overhauser et al., 1986bl. The somatic cell hybrid, HHW441, is derived from a patient with an interstitial deletion of 5p (46,XY,de1(5) (pter->pl5.l::pl6->qter) who is completely asymptomatic and shows no physical or mental abnormalities and has been described in detail elsewhere [Overhauser et al., 1986al. Genomic DNA was isolated from the somatic cell hybrids and digested to completion with EcoRI, which was subsequently electrophoresed through a 0.8% agarose gel and transferred to nylon membranes by capillary action as described [Overhauser et al., 1986al. Previously mapped DNA fragments were labeled with 32P using the random primer method [Feinberg and Vogelstein, 19841 and hybridized and washed using previously described methods [Overhauser et al., 1986al. RESULTS A set of 50 DNA fragments that had been previously mapped to 5p using a series of somatic cell hybrids containing different 5p deletions were used to screen the somatic cell hybrids derived from individual 11-4in this study (JH151 and JH162). Each probe was systematically hybridized to a Southern blot containing the new somatic cell hybrids. The examination of the hy-
bridization pattern of each probe to DNA from the various cell hybrids that lack different portions of 5p provided a straightforward method of mapping regionally the new somatic cell hybrids relative to the previously analyzed somatic cell hybrids and previously mapped DNA fragments. Representative autoradiographs from a subset of the DNA fragments screened are shown in Figure 4. The analysis confirmed the results of the highresolution cytogenetic analysis. In addition, the mapping allowed the comparison of the proximal and distal breakpoints relative to chromosome breakpoints in the other somatic cell hybrids, specifically with HHW441, a somatic cell hybrid derived from an individual who is asymptomatic. Probe pJON8E-A differentiated between the proximal deletion breakpoints of JH151, JH162, and HHW441 and showed that the HHW441 breakpoint was more proximal. Probe pJ0187H-A and others not shown were deleted in all 3 somatic cell hybrids, whereas pJOD124E-B and pJOD282E-C were deleted only in the cell hybrids JH151 and JH162. This confirmed that the 5p deletion described in this family has a more distal breakpoint than HHW441. The order of the different breakpoints relative to each other are shown in Figure 5 as well as the localizations of the DNA fragments dech,
pJ040E-A pJOL36H-A pJ ON8 E-A
pJOl87H-A pJODl24E-B pJOD282E-C pJOL17E-A
pJOB55E-A Fig. 4. Southern blot hybridization of 5p DNA fragments to digested somatic cell hybrid DNAs. The sample from which the DNA was obtained is shown above each lane. The hamster lane is DNA from the fusion parent from which all the somatic cell hybrids were obtained. The DNA fragment that was used for each hybridizationpattern seen is noted on the right.
Interstitial Deletion of 5p
359
ifestations may be due to the genetic background in which the chromosomal deletion is present and that the mental retardation and microcephaly are specific for the deletion. The grandmother did not have microcephaly and, by history, was less mentally retarded. It is unclear pJ040E-A 15.3 whether the Alzheimer disease is due to the 5p deletion. To date, no older cri-du-chat patients with deletions encompassing 5p13 have been described with Alzheimer-like symptoms. High-resolution studies were performed twice and showed no evidence of a balanced 15.2 translocation; mosaicism could not be ruled out in the grandmother. One possible reason for the reduced severity of the grandmother could be explained by genomic imprinting. The parental origin of the grandmother’s 15.1 I kHHW339 deletion could not be determined, but all the other afpJOL36H-A fected individuals obtained their 5p deletions from their pJON6E-A mother. If the grandmother’s deletion is of paternal origin, and differential methylation occurs in some of the genes deleted in this region, genomic imprinting could explain the variation in the clinical picture with this 5P specific deletion. 14 As shown in Figure 5, the deletion in HHW441, which pJ0167H-A is derived from an individual without clinical or mental abnormalities, is almost entirely contained within the present JH151 deletion present in JH151 and JH162. These findings study JH162 suggest that the clinical manifestations seen in the present family are the result of the additional chromosomal material from 5p13 that has been deleted. Thus a delepJOD124E-8 tion of 5p13 can result in mental retardation. Deletions in the short arm of chromosome 5 can lead to many different clinical manifestations. A terminal delepJOD282E-C tion of 5~15.33can result in the absence of clinical abnormalities, whereas a terminal deletion 5pter-> pJOL17E-A 13 JH141 5~15.31results in the cat-cry syndrome [Bengtsson et al., 19901. Deletions that include a critical portion of 5p15.2 result in the clinical manifestations of cri-duchat syndrome, whereas a deletion of most 5p14 results I I pJ0855E-A in the absence of any clinical manifestations [Overhauser et al., 1986al. Based on these case studies, deletions in 5p must be analyzed carefully before accurate prognosis can be determined. This is especially important with the advent of prenatal diagnosis. If a parent has a balanced translocation and offspring with an unbalanced karyotype have resulted, clinical prognosis of subsequent offspring with similar unbalanced karyoFig. 5. Physical location of the DNA fragments and the chromo- types can be determined [Overhauser et al., 19891.Howsome 5p deletions. The arrows represent terminal deletions, which remove the distal portion of 5p to the arrow. The brackets represent the ever, in most cases the occurrence of a 5p deletion is a de region of 5p lost in the interstitial deletions. The location of the DNA novo event with both parents having normal chromofragments relative to the somatic cell hybrids used in this study are somes [Niebuhr, 1978bl. It is clear from this report and shown on the right. others that prognosis in 5p deletions varies depending on the region deleted, and it will be necessary to analyze carefully any 5p deletions that are detected during prescribed in Figure 4, which were used to confirm the natal diagnosis so that a more accurate picture of the breakpoint order and location. outcome of the fetus can be portrayed to the parents.
I
~
1
r--JH132 1
DISCUSSION ACKNOWLEDGMENTS We describe a family in which an interstitial deletion J.O. was supported by a grant from the W.W. Smith of the short arm of chromosome 5 has been inherited in 3 Charitable Trust Foundation. generations. All affected individuals had some degree of REFERENCES mental retardation and had minor anomalies, whereas additional manifestations were unique to each affected Bengtsson U, McMahon J, Quarrel10,Rubenstein C, David K, Greenberg F, Wasmuth JJ (1990):Phenotypically normal carriers of unindividual. This suggests that the variable clinical man-
360
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balanced terminal deletions of 5p transmit the deletions to offspring who display growth and developmental delay. Am J Hum Genet 47:A208. Carlin ME (1990): The improved prognosis in cri-du-chat (5p-) syndrome. In Fraser WI (ed.): “Proc. 8th Cong Inter. Assoc. for the Scientific Study of Mental Deficiency.”Edinburgh, Scotland Blackwell. Dana S, Wasmuth JJ (1982b): Selective linkage disruption in humanChinese hamster cell hybrids: Deletion mapping of the leuS, h d , andchr genes on human chromosome 5. Mol Cell Biol2:1220-1228. Feinberg AP, Vogelstein B (1984): Addendum: A technique for radiolabelling DNA restriction fragments to high specific activity. Anal Biochem 137266-267. Niebuhr E (1978a):Cytologic observations in 35 individuals with a 5pkaryotype. Hum Genet 42:143-156. Niebuhr E (1978b):The cri du chat syndrome: Epidemiology, cytogenetics, and clinical features. Hum Genet 44:227-275. Overhauser J, Golbus MS, Schonberg SA, Wasmuth JJ (1986a): Molecular analysis of an unbalanced deletion of the short arm of chromosome 5 that produces no phenotype. Am J Hum Genet 39:l-10.
Overhauser J , Beaudet AL, Wasmuth JJ (1986b3: A fine structure physical map of the short arm of chromosome 5. Am J Hum Genet 39:562-572. Overhauser J, Bengtsson U, McMahon J , Ulm J, Butler MG, Santiago L,Wasmuth JJ (1989):Prenatal diagnosis and carrier detection of a cryptic translocation by using DNA markers from the short arm of chromosome 5. Am J Hum Genet 45:296-303. Walker JL, Blank CE, Smith BAM (1984): Interstitial deletion of the short arm of chromosome 5 in a mother and three children. J Med Genet 21:465-467. Wilkins LE (1981): The cri-du-chat syndrome: Population demographics, prometaphase chromosome analysis and karyotypephenotype correlations. Ph.D. dissertation, Medical College of Virginia, Richmond. Wilkins LE, Brown JA, Nance WE, Wolf B (1983): Clinical heterogeneity in 80 home-reared children with the cri-du-chat syndrome. J Pediatr 102528-533.
Clinical Phenotype and Molecular Analysis of a Three-Generation Family With an Interstitial Deletion of the Short Arm of Chromosome 5 L. D. Keppen, S. M. Gollin, D. Edwards, J. Sawyer, W.Wilson, and J. Overhauser Department of Pediatrics, University of South Dakota School of Medicine, Sioux Falls (L.D.K.); Department of Human Genetics, University of Pittsburgh, Pittsburgh (S.M.G.) and Department of Biochemistry and Molecular Biology, Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia (W.W., J.O.), Pennsylvania; and Departments of Pediatrics (D.E.) and Pathology (J.S.), UAMS, Little Rock, Arkansas (J.S.) do not seem to significantly alter the phenotype other than demonstrating a trend toward a greater degree of microcephaly,growth failure, and cognitive impairment [Wilkins, 1981; Wilkins et al., 1982; Carlin, 198911. Deletions that do not include the 5p15.2 critical region can result in varied clinical manifestations. An individual and several offspring with an interstitial deletion extending from 5p14 to 5p15.1 [de1(5)(5qter->5 p14::5p15.2->5pter)l had only microcephaly and mild mental retardation [Walker et al., 1984; Overhauser et al., 1986bl. In addition, 6 individuals in a 3-generation family with an interstitial deletion of 5p14[de1(5) (5qter->5p14::5~15.1>pterll were completely asymp0 1992 Wiley-Liss, Inc. tomatic and are mentally normal [Overhauser et al., 1986a1. The observation that 5p deletions can give rise to a wide range of clinical phenotypes demonstrates the KEY WORDS: chromosome 5, deletion, menneed to understand more clearly the role of each chromotal retardation, cri-du-chat somal region in the appearance of clinical manifestasyndrome tions and would allow for more accurate prognosis after prenatal diagnosis or the discovery ofa 5p deletion after birth. INTRODUCTION Here we describe a 3-generation family with an interDeletions in the short arm of chromosome 5 (5p) usu- stitial deletion of 5p that includes 5p14 and a portion of ally are associated with the cri-du-chat syndrome, char- 5p13. Clinical findings and comparison with previously acterized by a high-pitched, monochromatic cry; crani- published interstitial deletions are discussed. ofacial abnormalities, which include microcephaly, apparent hypertelorism, and micrognathia; a slowed CLINICAL REPORT rate of growth; and psychomotor retardation [Niebuhr, The family pedigree is shown in Figure 1.The propo197813; Carlin, 19891. Although the size of the deleted situs (111-1) was a small-for-gestational age infant with segment varies from patient to patient, there appears to microcephaly born to a 35-year old moderately retarded be a critical segment at 5p15.2 that is deleted in all mother. Birth weight was 1,630 g at 34 weeks of gestapatients with the cri-du-chat syndrome [Niebuhr, tion. At age 4 years, he has moderate mental retarda1978a; Overhauser et al., 1986bl. Larger deletions, tion, severe microcephaly,esotropia and hyperopia, midwhich include the loss of 5p material other than 5~15.2, face hypoplasia, and hypertonia (Fig. 2). His moderately retarded mother (11-4) has microcephaly, simple ears, micrognathia, scoliosis,and arachnodactyly. She can communicate only minimally, canReceived for publication December 9, 1991; revision received not read or write, and has an I& of 50. The maternal April 14, 1992. grandmother (1-1) was mildly mentally retarded and Address reprint requests to Joan Overhauser, Ph.D., Department of Biochemistry and Molecular Biology,Jefferson Institute of had kyphoscoliosis. The grandmother, mother, and propositus each had an Molecular Medicine, Thomas Jefferson University, 233 South 10th interstitial deletion of 5p. High-resolution chromosome Street, Philadelphia, PA 19107. We report on a %generation family with an interstitial deletion of the short a r m of chromosome 5. Varied manifestations were found among the affected individuals including microcephaly, hypertonia, and micrognathia; mental retardation was common to all affected individuals. High resolution chromosome analysis was interpreted as del(5) (pter->p14.3::~13.3->qter).Molecular comparison of the deletion in this family with individuals with other 5p deletions suggests that the clinical findings are due specifically to the chromosomal material deleted from 5p13.
0 1992 Wiley-Liss, Inc.
Interstitial Deletion of 5p
357
Y’
I
111
Fig. 1. Family pedigree. Left half of the symbol filled in represents cytogenetic confirmation of 5p deletion. Top right quarter filled in represents mental retardation. Bottom right quarter filled in represents mosaic trisomy 8.
analysis was interpreted as del(5)(pter->p14.3::~13.3-> qter) in the grandmother, mother, and propositus (Fig. 3). The grandmother has had 5 pregnancies. The oldest daughter (11-1) is more severely retarded than the mother of the propositus. She is said to have a facial appearance similar to that of the mother of the propositus and is in foster care. The second pregnancy resulted in a normal daughter (11-2).The third pregnancy was a spontaneous abortion at 4-5 months of gestation. The mother of the propositus was the result of the fourth pregnancy. The fifth pregnancy was a normal male who is 31 years old. It was difficult to evaluate the grandmother’s intelligence since she had Alzheimer disease. It was initially thought that she most likely had a balanced translocation since she did not have microcephaly and had reared her children, but cytogenetic abnormalities were not identified by repeated high resolution chromosome analysis. It was not possible t o get permission for a skin biopsy to rule out mosaicism. Other relatives were not available for evaluation. The mother later had another son (111-2) who had mosaic trisomy 8 in addition to deletion 5p. This was
Fig. 2. The propositus.
confirmed by fibroblast cell culture. Analysis of 30 trypsin-Giemsa banded metaphase cells from PHA-stimulated peripheral blood leukocyte cultures showed 7 cells (23%) with a 46,XY,de1(5)chromosome pattern and 23 cells (77%)with a 47,XY 8,de1(5)chromosome pattern. Analysis of 20 trypsin-Giemsa banded metaphase cells from tissue fibroblast cell cultures showed 8 cells (40%) with a 47,XY, + 8,de1(5) chromosome pattern and 12 cells (60%) with a 46,XY,de1(5) chromosome pattern. There was no evidence of mosaicism in the propositus by tissue fibroblast or lymphocyte cell culture. The son with del(5p) and mosaic trisomy 8 had severe
+
8
5 ---I
I
A
B
C
D
Fig. 3. Representative pairs of chromosome 5 from all affected relatives showing the interstitial deletion, del(5Kp13.3p14.3).A: 1-1,B:II-4;C:III-1;D:III-2.Arrows indicate position of the deletion. Dotted lines indicate centromere position.
358
Keppen et al.
mental retardation, microcephaly, downward slanting palpebral fissures, hyperopia and esotropia, thin, incompletely infolded ear helices, and 5th digit clinodactyly. He had hypertonia and has had poor weight gain. Based on the clinical picture of individuals with this 5p deletion, most of his abnormalities and the greater severity of mental retardation are due to mosaic trisomy 8. The microcephaly and mental retardation appear to be the only clinical features due to the 5p deletion. The presence of the mosaic trisomy 8 is unlikely to be related to the 5p deletion. Both sons were placed in foster care at the time of nursery discharge because the mother was unable to care for them. MATERIALS AND METHODS An Epstein-Barr virus-transformed cell line was established on individual 11-4, which was used to make somatic cell hybrid lines that specifically retained the deleted chromosome 5. The procedures used to make these human-Chinese hamster cell hybrids have been described in detail elsewhere [Dana and Wasmuth, 1982; Overhauser et al., 1986bl. The resulting somatic cell hybrids (JH151 and JH162) were analyzed using GTG banding to confirm the specific retention of the deleted chromosome 5. Additional somatic cell hybrids were used for comparison studies to map the chromosome breakpoints in the present family relative to other previously analyzed 5p deletions. Hybrid HHW213 contains the entire short arm of chromosome 5 and a small portion of the long arm [Dana and Wasmuth, 19821. Somatic cell hybrids HHW339, JH141, and JH132 are derived from patients with cri-du-chat syndrome and have terminal deletions at 5~15.1,5p13, and 5p13, respectively. The precise mapping of these breakpoints was determined using cytogenetic techniques (GTG-banding)as well as physical mapping using random DNA fragments from the short arm of chromosome 5 [Overhauser et al., 1986bl. The somatic cell hybrid, HHW441, is derived from a patient with an interstitial deletion of 5p (46,XY,de1(5) (pter->pl5.l::pl6->qter) who is completely asymptomatic and shows no physical or mental abnormalities and has been described in detail elsewhere [Overhauser et al., 1986al. Genomic DNA was isolated from the somatic cell hybrids and digested to completion with EcoRI, which was subsequently electrophoresed through a 0.8% agarose gel and transferred to nylon membranes by capillary action as described [Overhauser et al., 1986al. Previously mapped DNA fragments were labeled with 32P using the random primer method [Feinberg and Vogelstein, 19841 and hybridized and washed using previously described methods [Overhauser et al., 1986al. RESULTS A set of 50 DNA fragments that had been previously mapped to 5p using a series of somatic cell hybrids containing different 5p deletions were used to screen the somatic cell hybrids derived from individual 11-4in this study (JH151 and JH162). Each probe was systematically hybridized to a Southern blot containing the new somatic cell hybrids. The examination of the hy-
bridization pattern of each probe to DNA from the various cell hybrids that lack different portions of 5p provided a straightforward method of mapping regionally the new somatic cell hybrids relative to the previously analyzed somatic cell hybrids and previously mapped DNA fragments. Representative autoradiographs from a subset of the DNA fragments screened are shown in Figure 4. The analysis confirmed the results of the highresolution cytogenetic analysis. In addition, the mapping allowed the comparison of the proximal and distal breakpoints relative to chromosome breakpoints in the other somatic cell hybrids, specifically with HHW441, a somatic cell hybrid derived from an individual who is asymptomatic. Probe pJON8E-A differentiated between the proximal deletion breakpoints of JH151, JH162, and HHW441 and showed that the HHW441 breakpoint was more proximal. Probe pJ0187H-A and others not shown were deleted in all 3 somatic cell hybrids, whereas pJOD124E-B and pJOD282E-C were deleted only in the cell hybrids JH151 and JH162. This confirmed that the 5p deletion described in this family has a more distal breakpoint than HHW441. The order of the different breakpoints relative to each other are shown in Figure 5 as well as the localizations of the DNA fragments dech,
pJ040E-A pJOL36H-A pJ ON8 E-A
pJOl87H-A pJODl24E-B pJOD282E-C pJOL17E-A
pJOB55E-A Fig. 4. Southern blot hybridization of 5p DNA fragments to digested somatic cell hybrid DNAs. The sample from which the DNA was obtained is shown above each lane. The hamster lane is DNA from the fusion parent from which all the somatic cell hybrids were obtained. The DNA fragment that was used for each hybridizationpattern seen is noted on the right.
Interstitial Deletion of 5p
359
ifestations may be due to the genetic background in which the chromosomal deletion is present and that the mental retardation and microcephaly are specific for the deletion. The grandmother did not have microcephaly and, by history, was less mentally retarded. It is unclear pJ040E-A 15.3 whether the Alzheimer disease is due to the 5p deletion. To date, no older cri-du-chat patients with deletions encompassing 5p13 have been described with Alzheimer-like symptoms. High-resolution studies were performed twice and showed no evidence of a balanced 15.2 translocation; mosaicism could not be ruled out in the grandmother. One possible reason for the reduced severity of the grandmother could be explained by genomic imprinting. The parental origin of the grandmother’s 15.1 I kHHW339 deletion could not be determined, but all the other afpJOL36H-A fected individuals obtained their 5p deletions from their pJON6E-A mother. If the grandmother’s deletion is of paternal origin, and differential methylation occurs in some of the genes deleted in this region, genomic imprinting could explain the variation in the clinical picture with this 5P specific deletion. 14 As shown in Figure 5, the deletion in HHW441, which pJ0167H-A is derived from an individual without clinical or mental abnormalities, is almost entirely contained within the present JH151 deletion present in JH151 and JH162. These findings study JH162 suggest that the clinical manifestations seen in the present family are the result of the additional chromosomal material from 5p13 that has been deleted. Thus a delepJOD124E-8 tion of 5p13 can result in mental retardation. Deletions in the short arm of chromosome 5 can lead to many different clinical manifestations. A terminal delepJOD282E-C tion of 5~15.33can result in the absence of clinical abnormalities, whereas a terminal deletion 5pter-> pJOL17E-A 13 JH141 5~15.31results in the cat-cry syndrome [Bengtsson et al., 19901. Deletions that include a critical portion of 5p15.2 result in the clinical manifestations of cri-duchat syndrome, whereas a deletion of most 5p14 results I I pJ0855E-A in the absence of any clinical manifestations [Overhauser et al., 1986al. Based on these case studies, deletions in 5p must be analyzed carefully before accurate prognosis can be determined. This is especially important with the advent of prenatal diagnosis. If a parent has a balanced translocation and offspring with an unbalanced karyotype have resulted, clinical prognosis of subsequent offspring with similar unbalanced karyoFig. 5. Physical location of the DNA fragments and the chromo- types can be determined [Overhauser et al., 19891.Howsome 5p deletions. The arrows represent terminal deletions, which remove the distal portion of 5p to the arrow. The brackets represent the ever, in most cases the occurrence of a 5p deletion is a de region of 5p lost in the interstitial deletions. The location of the DNA novo event with both parents having normal chromofragments relative to the somatic cell hybrids used in this study are somes [Niebuhr, 1978bl. It is clear from this report and shown on the right. others that prognosis in 5p deletions varies depending on the region deleted, and it will be necessary to analyze carefully any 5p deletions that are detected during prescribed in Figure 4, which were used to confirm the natal diagnosis so that a more accurate picture of the breakpoint order and location. outcome of the fetus can be portrayed to the parents.
I
~
1
r--JH132 1
DISCUSSION ACKNOWLEDGMENTS We describe a family in which an interstitial deletion J.O. was supported by a grant from the W.W. Smith of the short arm of chromosome 5 has been inherited in 3 Charitable Trust Foundation. generations. All affected individuals had some degree of REFERENCES mental retardation and had minor anomalies, whereas additional manifestations were unique to each affected Bengtsson U, McMahon J, Quarrel10,Rubenstein C, David K, Greenberg F, Wasmuth JJ (1990):Phenotypically normal carriers of unindividual. This suggests that the variable clinical man-
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