Clinical, cytogenetic and molecular investigationsin three patients with WOE Hirschhorn syndrome Thies U, Back E, Wolff G, Schroeder-Kurth T, Hager H-D, Schroder K. Clinical, cytogenetic and molecular investigations in three patients with Wolf-Hirschhorn syndrome. Clin IGenet 1992: 42: 201-205.
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Clinical, cytogenetic and molecu1a.r studies were performed in three patients with Wolf-Hirschhorn syndrome (WHS). In all cases the altered chromosome 4 appeared to be the result of a de novo deletion. Cytogenetic investigations located the breakpoint at 4p15.3 and 4p13. With cytogenetic methods it was not possible to decide whether these deletions were terminal or interstitial. DNA methods also failed to define a distal breakpoint within the 4 ~ 1 6 . 3region which might have indicated an interstitial deletion. According to the literature, the paternal chromosome 4 is preferentially deleted in most patients with WHS. DNA analysis with polymorphic markers out of the 4p16.3 region revealed that in two of the cases reported here the deleted segment was of paternal and in one case of maternal origin.
Ulrike Thles', Elks Backz, Gerhard WOW, Traute Schroed~r-Kurth~, Hans-Dieter H a g d and Kirsten Schrider' 'Institut fur Humangenetik der Universital. Ghttingen, *Institut fur Humangenetik und Anthropologie der Universitat, heiburg i. Br. and 'lnstitut fur Humangenetik und Anlhropologie der Universital. Heidelberg. FRG
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Key words: chromosme abnormalities chromosome 4p deletin cytogenetic and molecular studies parental origin
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Dr. Ulrike Thies. lnstiiut fiir Humangenetik der Universitat. GoBlerstr. 12 d, W-3400 Gottingen. FRG Received 12 December 1991, revised 27 May, accepted for publication 9 June 1992
Wolf et al. (1965) and Hirschhiorn et al. (1965) described a hithertho unknowri syndrome characterized by prenatal and postnatal growth retardation, severe developmental retardation and congenital anomalies, which were .associated with a deletion of the distal part of ithe short arm of chromosome 4; this syndrome is now referred to as Wolf-Hirschhorn syndrome (WHS). More than 120 cases of WHS have been documented to date (Lurie et al. 1980, Schinzel 1984). WHS was found to result from cytogenetic anomalies which include bands 4p15 or 4p16 (Wilson et id. 1981). Patients with WHS were studied by Gusella et al. (1985) in order to chromosomally map the probe G8 (D4S10) linked to the Huntington disease region. The critical monosomic segment involved is located distally to D4S10 (McKeown et al. 1987, Anvret et al. 1991), which maps in 4~16.3.In most of the cases published so far, a paternal origin of the deleted chromosome can be demonstrated (Gusella et al. 1985, Olson & Magenis 1988, Quarrel1 et al. 1991). We have analysed three patients and their parents to determine the parental origin of the deleted chromosome using RFLP markers from 4~16.3.
Clinical reports and cytogenetics studies Case 1
This male patient was the first child born to healthy, unrelated parents, the mother being 26 and father 27 years old. Ultrasound study in the 6th month of pregnancy showed polyhydramnios and dystrophy. The first fetal movements were felt in the 25th week and spontaneous delivery occurred during the 42nd week of gestation. Apgar scores were 7/8/ 10. Birth measurements were: weight 2600 g, and length 51 cm. At birth the child was noted t o have muscular hypotonia, iris colobomata on the right eye, stenosis of the left nasolacrimal duct, microcornea, epicanthal fold, depressed nasal bridge, low-set ears, hypognathia, hemangioma on the left lower leg and a small umbilical hernia and hypospadias. Postnatal chromosome analysis of peripheral lymphocytes revealed a partial deletion of the short arm of chromosome 4. The RBA technique (Fig. la) and the GTG technique were applied to characterize the deletion. Either a terminal deletion: 46,XY, del (4) (p15.33) with loss of the bands p15.33 to pter, or an interstitial deletion: 46,XY, del (4) (p15.33 201
Thies et al.
Fig. I. Chromosome pairs no. 4 from Case 1 (a) RBA-technique. The deleted chromosome is on the left side. Chromosome pairs no. 4 from Case 2 (b) RBA technique. The deleted chromosome is on the left side. GTG-banding of chromosome 4 from Case 3 (c). Deleted chromosome on the left side.
p16.35) can be assumed. It is not possible to distinguish bet ween these possibilities by cytogenetics methods. The karyotypes of the parents were normal. X-ray determination of bone age at 3 months corresponded to a chronological age of 35th-36th week of gestation. The cardiological examinations were normal. The child had severe growth retardation at the age of 2 years: length 75 cm, weight 6.8 kg, head circumference 43 cm. The psychomotor development was that of a 9-month-old infant, but the child was able to walk by himself.
Case 2
This female patient was the 4th child born to healthy, non-consanguineous parents. The pregnancy was complicated by tachycardia from the 26th week and threatened abortion in the 6th month of gestation. Spontaneous delivery was in the 39th week. Apgar scores were 9/ 10110. Birth weight was 2100 g, length 46 cm, and head circumference 30.5 cm. At birth, the child showed muscular hypotonia, skin dimpling near the thumb, microcephaly, depressed nasal bridge, epicanthal folds, hypertelorism, low-set dysplastic ears, micrognathia, leftsided clubfoot, and congenital heart defect. Karyotyping of the newborn baby revealed an apparently identical deletion to that described in Case 1: 46,XX, del (4) (p15.33) or 46,XX, del (4) (p15.33 p16.35) (see Fig. lb). Both karyotypes of the parents were normal. At the age of 8 months, delayed psychomotor development and growth failure were evident. At this time she developed epileptic seizures which required continuous treatment. The heart defect was diagnosed as VSD and pulmonary stenosis. At 202
the age of 4 years she is severely mentally and physically retarded (length 85 cm, weight 7.6 kg), She cannot walk and is still not speaking but is crawling. When spoken to she does not comprehend the meaning but shows meaningful reactions while playing. Case 3
This boy is the first child of healthy parents. At the time of his delivery, the mother was 26 and the father 28 years old. The pregnancy was complicated by severe proportional growth retardation of the fetus during the 3rd trimester and by hydramnios. The ultrasound investigation revealed no gross malformations. Amniocentesis was done for further obstetric management in the 31st week of pregnancy. The fetal karyotype was 46,XY del (4p). The breakpoint appeared to be at p13 (Fig. lc). Both parents have normal karyotypes. The symptoms of the child are muscular hypertonia, microcephaly, ocular hypertelorism, epicanthal folds, prominent glabella, no cleft lip or palate, down-turned fish-like mouth, hypospadias and cryptorchidism. DNA analysis
DNA from peripheral blood lymphocytes or Epstein-Ban virus-transformed lymphocyte cell lines (Neizel 1986) from the patients were tested with polymorphic DNA probes of the 4p 16.3 region (see Table 1). Agarose gel electrophoresis, Southern blot analysis and hybridizations were camed out according to standard procedures. Paternity was confirmed using the oligonucleotide probe (CAC)5 for DNA fingerprinting (Schafer et al. 1988).
Chromosomal deletion in WHS Table 1. Probes of the 4p16.3 region revealing parental allele loss in three WHS patients
Locus
Probes
enzyme
Bgl II EcoR I Hind 111 site 1 Hind 111 site 2 D4SlO pcos 5.52 Msp I Bgl I1 D4S81 RB1.6 sac I D4S126 p309 Acc I D4S95 p674 Mbo I Taq I Taq I 04543 s1.5 Hind 111 54.9 41 2R3 Pvu II D4S90 D5
D4S10 D4S10 D4S10
Probes revealing hornozygousity Case1
Case2
Case3
pTvZ0
pTv20 pK083 pK082
pK083
pcos d52 p309 p674
sl.5 2R3
Loci listed in order from the most proximal (D4S10) to the most telomeric (D4S90). Each of the probes is described in HGM 11.
Results
The Southern blot analysis from Case 1 showed paternal allele loss when probed with D4S10 (pcos 5.52/Msp I). The mother turned out to be heterozygous, the father was homozygous for the major allele, while the propositus was hemizygous for the minor allele. Another marker D4S126 (p309) confirmed the result of paternal allele loss (see Fig.
in cases with deletions, ranging from (4) (pter-p16) to (4) (pter-p13) (Schinzel 1984). The genes responsible for WHS are located more distally than previous cytogenetic observations have suggested. Clinical and cytogenetic investigation of a large (4;8) translocation family with Wolf syndrome by Tranebjaerg et al. (1984) demonstrated that loss of the terminal segment of 4p (4~16.3)seems sufficient to produce the clinical entity of Wolf syndrome. Molecular studies of the major patient of this translocation family proved that D4S 10 locus is hemizygous in the affected child, supporting the 4p16.3 localization of this polymorphic marker (MacDonald et al. 1987). Dodge & Read (1989) localized the breakpoint in one WHS patient between D4SiO and D4S43. McKeown et al. (1987) and Anvret et al. (1991) determined the breakpoint to be distal to D4S10. Altherr et al. (1991) reported a girl with clinical features of WHS whose mother carried a subtle translocation between chromosome 4 and 9, which resulted in an unbalanced form in the child. Molecular studies using the probes D4S95 and D4S97 revealed a deletion for the distal 4-5 million bp of 4 p, resulting in the loss of the maternal haplotype. Assuming terminal deletions, the present cytogenetic investigations located the breakpoint proxi-
2).
DNA typing in Case 2 revealed that the paternal chromosome was also involved in the deletion. Three marker/enzyme constellations turned out to be informative. The most distal marker 2R3/Hind I11 defined by the D4S141 locus, probe sl.S/TaqI from the D4S43 locus in the c(entra1part of the 4p16.3 region (see Fig. 3) and the most proximal marker pTV 20 (D4S10) documented loss of paternal aIleles in the patient. In Case 3 the maternal chromosome 4 is deleted. Two marker/enzyme constellations were informative: the proximal marker (pK083/EcoRI) (D4S10) and the central marker p674/AccI (D4S95). Using pK083/EcoRI the child turned out to be hemizygous for the major allele, while the mother was homozygous for the minor allele and the father homozygous for the major allele (see Fig. 4). At p674/AccI, both the biallelic anti the VNTR polymorphism indicated that the maternal allele was lost.
p309(D4sl28)ISac I C f m
allele 1 dele 2 Iu 1
h)
-. -. I
N I
h)
Discussion
The patients described here showed the typical clinical features of the WHS. From familial cases it appears that the clinical picture and the degree of pre- and postnatal development ilre fairly constant
Fig. 2. Case 1: Hybridization pattern of D4S126 (p309/SacI) digests of genomic DNA of the patient (c), his father (f) and his mother (m). The marker shows loss of the paternal allele in the patient.
203
This et al. ma1 to the 4p16.3 region (4~15.3(Case 1 and 2) and 4p13 (Case 3). To localize the distal breakpoint of a possible interstitial deletion, 4p16.3 probes were used. One patient (Case 2) could be shown to be monosomic at least up to 325 kb to the telomere by probe 2R3, a subclone of D4S90. The parental origins of several de n o w chromosomal abnormalities have been defined by molecular studies. Deletions in Prader-Willi syndrome (Butler et al. 1986, Nicholls et al. 1989) or Cri-duchat syndrome (Overhauser et al. 1989) have been reported to be preferentially of paternal origin, whereas in several cases with Angelman syndrome (Magenis et al. 1990, Williams et al. 1990) and a single case of Langer-Giedion syndrome (Liidecke et al. 1989) the deletions were of maternal origin. So far, paternal origin of the deletions has been reported in 10 of 11 informative cases of WHS (Gusella et al. 1985, Olson & Magenis 1988, Quarre11 et al. 1991, Anvret et al. 1991). To the best of our knowledge, Case 3 in this report is the second example of WHS resulting from a maternal deletion. Thus from 14 patients studied by DNA haplotyping, so far two deletions have been of maternal origin. These results could indicate a preferential paternal origin of the deleted chromosome in WHS. A strong bias toward paternal origin of de n o w structural chromosome rearrangements (84.4%)
S1.5(D4S43)/Taq I f m
pK083(D4S1O)/EcoR I
h
m
f
C
*'-=* allele 1 allele 2
.._.... ._.____................ ........ .....-...........-...._. .._._-.... ~
-..
A
I
*
Iu
A
~
Iu I
Iu
4 I
-..
Fig. 4. Case 3: Southern blot of genomic DNA digested with EcoRl and hybridised to subclone pK083 of the D4SIO locus. showing a loss of the maternal allele. c=patient, f=father, m = mother, h = control person for heterozygosity.
was reported by Olson & Magenis (1988) and Chamberlin & Magenis (1980) (76.5Y0). Thus 12 out of 14 4p-deletions of paternal origin is entirely consistent with the observed ratio in other structural rearrangements. Further molecular analysis of WHS patients should give more information about the parental origin and a more detailed localisation of the genes involved in this syndrome.
C
allele 1 Note added in proof
allele 2
In a recently published study the paternal origin of the de novo deleted chromosome 4 in five WHS patients was assessed by Tupler et al. (1 992).
Acknowledgements h) I
10
* -L
-L
* -L
Fig. 3. Case 2: Southern blot analysis of DNA from the patient (c) her mother (m) and her father (f) showing a loss of the paternal allele when probed with D4S43 (sl.S/TaqI).
204
We would like to express our thanks to the parents for allowing us to obtain blood samples from their children. We thank D. Niederhoff from the Universitats-Kinderklinik, Freiburg for clinical data of patients 1 and 2, J. Zimmer and H. Schulz for technical assistance with lymphoblast cultures and A. Czarny for typing the manuscript. We thank L. R. Carlock, F. Collins, J. F. Gusella, P. S. Harper, M. R. Hayden, for providing the probes used in this study and W.Engel for support and helpful discussion.
Chromosomal deletion in WHS
References Altherr MR, Bengtsson U, Elder FFB, Ledbetter DH, Wasmuth J, McDonald ME, Gusella IF, Greeriberg F. Molecular confirmation of Wolf-Hirschhorn syndrome with a subtle translocation of chromosome 4. Am J Hum Genet 1991: 49: 1235-1 242. Anvret M, Nordenskjiild M, Stolpe L, Johannson L, BrondumNielsen K. Molecular analysis of 4p deletion associated with Wolf-Hirschhorn syndrome moving the “critical segment’’ towards the telomere. Hum Genet 1991: 86: 481-483. Butler MG, Meaney FJ, Palmer CG. Clinical and cytogenetic survey of 39 individuals with Prader-Lambert-Willi syndrome. Am J Med Genet 1986: 23: 793-809. Chamberlin J, Magenis RE. Paternal origin of the de now chromosome rearrangements. Hum Genet 1980: 53: 343-347. Dodge A. Read AP. Definition of a chiromosomal breakpoint near to the Huntington’s disease locus. J Med Genet 1989: 26: 216215. Gusella JF, Tanti RE, Bader PI, Phelan MC, Stevenson R. Hayden MR, Hofrnan KJ, Faryniarx AG, Gibbons K. Deletion of Huntington’s disease - linked G8 (D4S10) locus in Wolf-Hirschhorn syndrome. Nature il985: 318: 75-78. Hirschhorn K, Cooper HL, Firschein IL. Deletion of short arms of chromosome 4-5 in a child with defects of midline fusion. Humangenetik 1965: I: 479-482. Liidecke HJ, Burdieck R, Senger G. Claussen U, Passarge E. Horsthemke B. Maternal origin of a de novo chromosome 8 deletion in a patient with Langer-Giedion syndrome. Hum Genet 1989: 82: 327-329. Lurie IW. Lazjuk GI, Ussova I, Presman EB, Gurevich DB. The Wolf-Hirschhorn syndrome: 1. Genetics. Clin Genet 1980: 17: 315-385. MacDonald ME, Anderson MA. Gilliam TC, Tranebjaerg L. Carpenteer NJ, Magenis E. Hayden MR, Healey ST. Bonner TI, Gusella JF. A somatic cell hybrid panel for localizing DNA segments near the Huntingon’s ‘diseasegene. Genomics 1987: I : 29-34. Magenis RE, Toth-Fejel S, Allen U,Black M, Brown MG. Budden S. Cohen R, Friedmann JM. Kalousek D, Zonana J, Lacy D, La Franchi S, Lahr M, klacFarlane J. Williams GPS. Comparison of the 15 q deletions in Prader-Willi and Angelman syndromes: specific regions, extent of deletions, parental origin and clinical consequences. Am J Med Genet 1990: 35: 333-349. McKeown C. Read AP. Dodge A. Steck.0 0,Mercer A, Harris R. Wolf-Hirschhorn locus is distal to D4SIO on short arm of chromosome 4. J Med Genet 1987: 24: 410-412.
Neizel H. A routine method for the establishment of permanent growing lymphobastoid cell lines. Hum Genet 1986: 73: 320-326. Nicholls RD, Knoll JH, Glatt K, Hersch JH, Brewster TD, Graham JM, Wurster-Hill D,Wharton R, Latt SA. Restriction fragment length polymorphisms within proximal 15 q and their use in molecular cytogenetic and the Prader-Wilti syndrome. Am J Med Genet 1989: 33: 6677. Olson SB, Magenis RE. Preferential paternal origin of the de n o w structural chromosome rearrangements. In: Daniel A ed. The cytogenetics of mammalian autosomal rearrangements. New York Alan R. Liss, 1988: 583-599. Overhauser J, Lee-Chen GJ, McMahn J, Wasmuth J, Carlin ME, Oberlender S, Niebuhr E. Paternal inheritance of the deleted chromosome 5 in cri du chat syndrome patients. Am J Hum Genet 1989: S5: (suppl): 85 A. Quarrel1 OWJ, Snell RG, Curtis MA, Roberts SH, Harper PS, Shaw DJ. Paternal origin of the chromosomal deletion resulting in Wolf-Hirschhorn syndrome. J Med Genet 1991: 28: 256259. Schlfer R, Zischler H. Epplen J. (CAC)s a very informative oligo-nucleotide probe for DNA fingerprinting. Nucleic Acids Res 1988: 11: 5196. Schinzel A ed. Catalogue of unbalanced chromosome aberrations in man. Berlin, New York: Walter de Gruyter, 1984: 161-1 64. Tranebjaerg L, Petersen A, Hove K, Rehder H, Mikkelsen M. Clinical and cytogenetics studies in a large (4;8) translocation family with pre- and postnatal Wolf syndrome. Am J Med Genet 1984: 27: 224-229. Tupler R, Bortotto L, Buhler EM, AIkan M, Malik NJ. BoschAl Jodova N, Memo L, Maraxhio P. Paternal origin of the de nuvo deleted chromosome 4 in Wolf-Hirschhorn syndrome. J Med Genet 1992: 29: 53-55. Williams CA. Zori RT, Stone JW, Gray BA, Cantu ES, Ostrer H. Maternal origin of 15qll-13 deletion in Angelmann syndrome suggests a role for genomic imprinting. Am J Med Genet 1990: 35: 350-353. Wilson MG, Towner JW, Coflin GS. Ebbin AJ, Siris E, Brager P. Genetic and clinical studies in 13 patients with the WolfHirschhorn syndrome [del(4p)]. Hum Genet 1981: 59: 297-307. Wolf U, Reinwein H, Porsch R, Schriiter R, Baitsch H. Defizienz an den kunen Armen eines Chromosomes Nr. 4. Humangenetik 1965: 1: 397413.
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Clinical, cytogenetic and molecu1a.r studies were performed in three patients with Wolf-Hirschhorn syndrome (WHS). In all cases the altered chromosome 4 appeared to be the result of a de novo deletion. Cytogenetic investigations located the breakpoint at 4p15.3 and 4p13. With cytogenetic methods it was not possible to decide whether these deletions were terminal or interstitial. DNA methods also failed to define a distal breakpoint within the 4 ~ 1 6 . 3region which might have indicated an interstitial deletion. According to the literature, the paternal chromosome 4 is preferentially deleted in most patients with WHS. DNA analysis with polymorphic markers out of the 4p16.3 region revealed that in two of the cases reported here the deleted segment was of paternal and in one case of maternal origin.
Ulrike Thles', Elks Backz, Gerhard WOW, Traute Schroed~r-Kurth~, Hans-Dieter H a g d and Kirsten Schrider' 'Institut fur Humangenetik der Universital. Ghttingen, *Institut fur Humangenetik und Anthropologie der Universitat, heiburg i. Br. and 'lnstitut fur Humangenetik und Anlhropologie der Universital. Heidelberg. FRG
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Key words: chromosme abnormalities chromosome 4p deletin cytogenetic and molecular studies parental origin
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Dr. Ulrike Thies. lnstiiut fiir Humangenetik der Universitat. GoBlerstr. 12 d, W-3400 Gottingen. FRG Received 12 December 1991, revised 27 May, accepted for publication 9 June 1992
Wolf et al. (1965) and Hirschhiorn et al. (1965) described a hithertho unknowri syndrome characterized by prenatal and postnatal growth retardation, severe developmental retardation and congenital anomalies, which were .associated with a deletion of the distal part of ithe short arm of chromosome 4; this syndrome is now referred to as Wolf-Hirschhorn syndrome (WHS). More than 120 cases of WHS have been documented to date (Lurie et al. 1980, Schinzel 1984). WHS was found to result from cytogenetic anomalies which include bands 4p15 or 4p16 (Wilson et id. 1981). Patients with WHS were studied by Gusella et al. (1985) in order to chromosomally map the probe G8 (D4S10) linked to the Huntington disease region. The critical monosomic segment involved is located distally to D4S10 (McKeown et al. 1987, Anvret et al. 1991), which maps in 4~16.3.In most of the cases published so far, a paternal origin of the deleted chromosome can be demonstrated (Gusella et al. 1985, Olson & Magenis 1988, Quarrel1 et al. 1991). We have analysed three patients and their parents to determine the parental origin of the deleted chromosome using RFLP markers from 4~16.3.
Clinical reports and cytogenetics studies Case 1
This male patient was the first child born to healthy, unrelated parents, the mother being 26 and father 27 years old. Ultrasound study in the 6th month of pregnancy showed polyhydramnios and dystrophy. The first fetal movements were felt in the 25th week and spontaneous delivery occurred during the 42nd week of gestation. Apgar scores were 7/8/ 10. Birth measurements were: weight 2600 g, and length 51 cm. At birth the child was noted t o have muscular hypotonia, iris colobomata on the right eye, stenosis of the left nasolacrimal duct, microcornea, epicanthal fold, depressed nasal bridge, low-set ears, hypognathia, hemangioma on the left lower leg and a small umbilical hernia and hypospadias. Postnatal chromosome analysis of peripheral lymphocytes revealed a partial deletion of the short arm of chromosome 4. The RBA technique (Fig. la) and the GTG technique were applied to characterize the deletion. Either a terminal deletion: 46,XY, del (4) (p15.33) with loss of the bands p15.33 to pter, or an interstitial deletion: 46,XY, del (4) (p15.33 201
Thies et al.
Fig. I. Chromosome pairs no. 4 from Case 1 (a) RBA-technique. The deleted chromosome is on the left side. Chromosome pairs no. 4 from Case 2 (b) RBA technique. The deleted chromosome is on the left side. GTG-banding of chromosome 4 from Case 3 (c). Deleted chromosome on the left side.
p16.35) can be assumed. It is not possible to distinguish bet ween these possibilities by cytogenetics methods. The karyotypes of the parents were normal. X-ray determination of bone age at 3 months corresponded to a chronological age of 35th-36th week of gestation. The cardiological examinations were normal. The child had severe growth retardation at the age of 2 years: length 75 cm, weight 6.8 kg, head circumference 43 cm. The psychomotor development was that of a 9-month-old infant, but the child was able to walk by himself.
Case 2
This female patient was the 4th child born to healthy, non-consanguineous parents. The pregnancy was complicated by tachycardia from the 26th week and threatened abortion in the 6th month of gestation. Spontaneous delivery was in the 39th week. Apgar scores were 9/ 10110. Birth weight was 2100 g, length 46 cm, and head circumference 30.5 cm. At birth, the child showed muscular hypotonia, skin dimpling near the thumb, microcephaly, depressed nasal bridge, epicanthal folds, hypertelorism, low-set dysplastic ears, micrognathia, leftsided clubfoot, and congenital heart defect. Karyotyping of the newborn baby revealed an apparently identical deletion to that described in Case 1: 46,XX, del (4) (p15.33) or 46,XX, del (4) (p15.33 p16.35) (see Fig. lb). Both karyotypes of the parents were normal. At the age of 8 months, delayed psychomotor development and growth failure were evident. At this time she developed epileptic seizures which required continuous treatment. The heart defect was diagnosed as VSD and pulmonary stenosis. At 202
the age of 4 years she is severely mentally and physically retarded (length 85 cm, weight 7.6 kg), She cannot walk and is still not speaking but is crawling. When spoken to she does not comprehend the meaning but shows meaningful reactions while playing. Case 3
This boy is the first child of healthy parents. At the time of his delivery, the mother was 26 and the father 28 years old. The pregnancy was complicated by severe proportional growth retardation of the fetus during the 3rd trimester and by hydramnios. The ultrasound investigation revealed no gross malformations. Amniocentesis was done for further obstetric management in the 31st week of pregnancy. The fetal karyotype was 46,XY del (4p). The breakpoint appeared to be at p13 (Fig. lc). Both parents have normal karyotypes. The symptoms of the child are muscular hypertonia, microcephaly, ocular hypertelorism, epicanthal folds, prominent glabella, no cleft lip or palate, down-turned fish-like mouth, hypospadias and cryptorchidism. DNA analysis
DNA from peripheral blood lymphocytes or Epstein-Ban virus-transformed lymphocyte cell lines (Neizel 1986) from the patients were tested with polymorphic DNA probes of the 4p 16.3 region (see Table 1). Agarose gel electrophoresis, Southern blot analysis and hybridizations were camed out according to standard procedures. Paternity was confirmed using the oligonucleotide probe (CAC)5 for DNA fingerprinting (Schafer et al. 1988).
Chromosomal deletion in WHS Table 1. Probes of the 4p16.3 region revealing parental allele loss in three WHS patients
Locus
Probes
enzyme
Bgl II EcoR I Hind 111 site 1 Hind 111 site 2 D4SlO pcos 5.52 Msp I Bgl I1 D4S81 RB1.6 sac I D4S126 p309 Acc I D4S95 p674 Mbo I Taq I Taq I 04543 s1.5 Hind 111 54.9 41 2R3 Pvu II D4S90 D5
D4S10 D4S10 D4S10
Probes revealing hornozygousity Case1
Case2
Case3
pTvZ0
pTv20 pK083 pK082
pK083
pcos d52 p309 p674
sl.5 2R3
Loci listed in order from the most proximal (D4S10) to the most telomeric (D4S90). Each of the probes is described in HGM 11.
Results
The Southern blot analysis from Case 1 showed paternal allele loss when probed with D4S10 (pcos 5.52/Msp I). The mother turned out to be heterozygous, the father was homozygous for the major allele, while the propositus was hemizygous for the minor allele. Another marker D4S126 (p309) confirmed the result of paternal allele loss (see Fig.
in cases with deletions, ranging from (4) (pter-p16) to (4) (pter-p13) (Schinzel 1984). The genes responsible for WHS are located more distally than previous cytogenetic observations have suggested. Clinical and cytogenetic investigation of a large (4;8) translocation family with Wolf syndrome by Tranebjaerg et al. (1984) demonstrated that loss of the terminal segment of 4p (4~16.3)seems sufficient to produce the clinical entity of Wolf syndrome. Molecular studies of the major patient of this translocation family proved that D4S 10 locus is hemizygous in the affected child, supporting the 4p16.3 localization of this polymorphic marker (MacDonald et al. 1987). Dodge & Read (1989) localized the breakpoint in one WHS patient between D4SiO and D4S43. McKeown et al. (1987) and Anvret et al. (1991) determined the breakpoint to be distal to D4S10. Altherr et al. (1991) reported a girl with clinical features of WHS whose mother carried a subtle translocation between chromosome 4 and 9, which resulted in an unbalanced form in the child. Molecular studies using the probes D4S95 and D4S97 revealed a deletion for the distal 4-5 million bp of 4 p, resulting in the loss of the maternal haplotype. Assuming terminal deletions, the present cytogenetic investigations located the breakpoint proxi-
2).
DNA typing in Case 2 revealed that the paternal chromosome was also involved in the deletion. Three marker/enzyme constellations turned out to be informative. The most distal marker 2R3/Hind I11 defined by the D4S141 locus, probe sl.S/TaqI from the D4S43 locus in the c(entra1part of the 4p16.3 region (see Fig. 3) and the most proximal marker pTV 20 (D4S10) documented loss of paternal aIleles in the patient. In Case 3 the maternal chromosome 4 is deleted. Two marker/enzyme constellations were informative: the proximal marker (pK083/EcoRI) (D4S10) and the central marker p674/AccI (D4S95). Using pK083/EcoRI the child turned out to be hemizygous for the major allele, while the mother was homozygous for the minor allele and the father homozygous for the major allele (see Fig. 4). At p674/AccI, both the biallelic anti the VNTR polymorphism indicated that the maternal allele was lost.
p309(D4sl28)ISac I C f m
allele 1 dele 2 Iu 1
h)
-. -. I
N I
h)
Discussion
The patients described here showed the typical clinical features of the WHS. From familial cases it appears that the clinical picture and the degree of pre- and postnatal development ilre fairly constant
Fig. 2. Case 1: Hybridization pattern of D4S126 (p309/SacI) digests of genomic DNA of the patient (c), his father (f) and his mother (m). The marker shows loss of the paternal allele in the patient.
203
This et al. ma1 to the 4p16.3 region (4~15.3(Case 1 and 2) and 4p13 (Case 3). To localize the distal breakpoint of a possible interstitial deletion, 4p16.3 probes were used. One patient (Case 2) could be shown to be monosomic at least up to 325 kb to the telomere by probe 2R3, a subclone of D4S90. The parental origins of several de n o w chromosomal abnormalities have been defined by molecular studies. Deletions in Prader-Willi syndrome (Butler et al. 1986, Nicholls et al. 1989) or Cri-duchat syndrome (Overhauser et al. 1989) have been reported to be preferentially of paternal origin, whereas in several cases with Angelman syndrome (Magenis et al. 1990, Williams et al. 1990) and a single case of Langer-Giedion syndrome (Liidecke et al. 1989) the deletions were of maternal origin. So far, paternal origin of the deletions has been reported in 10 of 11 informative cases of WHS (Gusella et al. 1985, Olson & Magenis 1988, Quarre11 et al. 1991, Anvret et al. 1991). To the best of our knowledge, Case 3 in this report is the second example of WHS resulting from a maternal deletion. Thus from 14 patients studied by DNA haplotyping, so far two deletions have been of maternal origin. These results could indicate a preferential paternal origin of the deleted chromosome in WHS. A strong bias toward paternal origin of de n o w structural chromosome rearrangements (84.4%)
S1.5(D4S43)/Taq I f m
pK083(D4S1O)/EcoR I
h
m
f
C
*'-=* allele 1 allele 2
.._.... ._.____................ ........ .....-...........-...._. .._._-.... ~
-..
A
I
*
Iu
A
~
Iu I
Iu
4 I
-..
Fig. 4. Case 3: Southern blot of genomic DNA digested with EcoRl and hybridised to subclone pK083 of the D4SIO locus. showing a loss of the maternal allele. c=patient, f=father, m = mother, h = control person for heterozygosity.
was reported by Olson & Magenis (1988) and Chamberlin & Magenis (1980) (76.5Y0). Thus 12 out of 14 4p-deletions of paternal origin is entirely consistent with the observed ratio in other structural rearrangements. Further molecular analysis of WHS patients should give more information about the parental origin and a more detailed localisation of the genes involved in this syndrome.
C
allele 1 Note added in proof
allele 2
In a recently published study the paternal origin of the de novo deleted chromosome 4 in five WHS patients was assessed by Tupler et al. (1 992).
Acknowledgements h) I
10
* -L
-L
* -L
Fig. 3. Case 2: Southern blot analysis of DNA from the patient (c) her mother (m) and her father (f) showing a loss of the paternal allele when probed with D4S43 (sl.S/TaqI).
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We would like to express our thanks to the parents for allowing us to obtain blood samples from their children. We thank D. Niederhoff from the Universitats-Kinderklinik, Freiburg for clinical data of patients 1 and 2, J. Zimmer and H. Schulz for technical assistance with lymphoblast cultures and A. Czarny for typing the manuscript. We thank L. R. Carlock, F. Collins, J. F. Gusella, P. S. Harper, M. R. Hayden, for providing the probes used in this study and W.Engel for support and helpful discussion.
Chromosomal deletion in WHS
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