Chromosome 22 microdeletion by F.I.S.H. in isolated congenital heart disease
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《美国医学杂志》
1 Genetic Research Centre, National Institute for Research in Reproductive Health (ICMR), J.M. Street, Parel, Mumbai, India
2 Department of Cardiology, Cardiovascular Thoracic Center, King Edward Memorial (KEM) Hospital, Parel, Mumbai, India
Abstract
Objective. To analyze the frequency of del22q11.2 in non-syndromic CHDs using classical cytogenetics and Fluorescence In Situ Hybridization (FISH) technique in Indian population. Methods. 105 prospective cases which included 6 families with isolated, non-syndromic cardiac defects were analyzed clinically by a cardiologist and a geneticist. The cases were then subjected to karyotypic (classical cytogenetics) as well as FISH analysis. The efficacy of FISH technique was compared with inference drawn from classical cytogenetics. Results. Karyotypic analysis of all the 105 patients revealed a normal chromosomal complement. Microdeletion 22q11.2 was observed in six patients (5.71%) by FISH studies. FISH studies were also performed on the parents of these six patients who revealed a normal chromosome 22. No correlation was found between clinical features (mild or unspecific) with 22q11.2 microdeletion. Conclusion. The testing for microdeletion 22q11.2 in isolated non-syndromic patients using FISH technique is mandatory even when mild/unspecific extracardiac abnormalities are seen in the patients.
Keywords: FISH; India; Isolated congenital heart disease (CHD); Microdeletion 22q11.2
Congenital heart diseases (CHDs) are the most common of all birth defects and are the leading cause of mortality in the first year of life with a prevalence of 1% in live births and 10% in spontaneously aborted fetus.[1] One of the most common syndromes associated with CHDs is the 22q11.2 deletion (del22q11.2) syndrome, which has an estimated prevalence of approximately 1 in 4000 live births [2], [3] of which 90% of cases are sporadic.[4] CHDs are a multifactorial defects caused by (a) 90% multifactorial disorders (b) chromosomal disorders and single gene disorders constituting 8% and (c) 2% of environmental teratogens.[5] They are the most common single group of congenital abnormalities accounting for about 30% of the total abnormalities.[6] In most patients, CHDs occur as an isolated malformation, but about 33% have associated anomalies [7].
The various conditions associated with del22q11 include DiGeorge anomaly (DG), Velocardiofacial syndrome (VCFS), Conotruncal anomaly face syndrome (CTAFS), isolated conotruncal heart defects (CTHDs) and Cayler cardiofacial syndrome.[8],[9],[10],[11] The abnormalities that are already known to be associated with this deletion affect multiple body systems, including cranofacial and otolaryngeal structures, cardiovascular, endocrine, nervous, musculoskeletal and immune processes.[12] As a result of their common genetic origin and their phenotypic overlap the syndromes were collectively referred to as "CATCH 22" (Cardiac abnormality, Abnormal facies, Thymic hypoplasia, Cleft palate, Hypocalcemia and del22q11.2.[11],[13] The cardiac defects commonly seen in del22q11.2 syndrome are derived from conotruncus, the embryonic aortic arches and the ventricular septum which are Tetralogy of Fallot (TOF), Ventricular Septal Defect (VSD), Atrial Septal Defect (ASD), Truncus Arteriosus (TA), and Interrupted Aortic Arch (IAA). [14]
Previous studies on the presence of 22q11.2 microdeletion in patients with isolated CHDs have been reported since the early 1990s.[9],[15],[16] However, such studies have not been taken up in India till date. The present investigation was initiated in order to look into the issue of microdeletion 22q11.2 in a non-selected patient group in Indian population and to develop genetic guidelines for microdeletion testing which will help in future evaluation of patients with conotruncal defects. Two similar studies in unselected patients with CHD have been reported [17],[18] and ours is the third in the series.
Materials and methods
In the present study, a total of 105 subjects (consecutive, non-selected) were enrolled prospectively from OPD of Cardio Vascular Thoracic Center (CVTC), KEM hospital as well as from Genetic Research Center Clinic (NIRRH). The subjects belonged to the age group ranging from 24 days to 24 years. The cardiologist from CVTC clinically confirmed the CHD by ECG, 2D Echocardiography and Colour Doppler study in all subjects included in the present investigation. The clinical geneticist ascertained the clinical data, which included weight, height, type of CHD, dysmorphisms, extra cardiac malformations (if any) and psychomotor development.
Peripheral venous blood was obtained from each patient for karyotypic and Fluorescence in Situ Hybridization (FISH) analysis, with the consent of the family members. Chromosomal analysis was carried out on peripheral blood lymphocyte culture using standard protocol with suitable modifications (Seabright 1971). [19] G-banded metaphase chromosomes were screened at 550-band level. The chromosomes were analyzed and karyotyped according to the International System for Human Cytogenetic Nomenclature (ISCN 1995). [20]
Fluorescence in situ Hybridization (FISH) was performed on metaphase chromosome spreads using the VYSIS LSI DiGeorge syndrome probe which maps to the TUPLE1 region i.e. 22q11.2 (spectrum orange) combined with 22q11.3 (spectrum green) region control probe according to manufacturers instructions.
In short, slides were treated with 70% acetic acid for 15 minutes at room temperature, then dehydrated in a series of alcohol grades 70%, 85% 100% for 3 minutes each, then treated with pepsin (1mg/ml) final stock for 3 minutes at 37 oC. The slides were then washed in 1XPBS for 5 minutes, dehydrated in a series of alcohol grades for 3 minutes each and air-dried. Probe mixture was prepared according to manufacturers instructions. The probe mixture was then applied to the slide, coverslip placed, sealed with rubber cement and slide was subjected to co-denaturation on a hot plate for 4 minutes at 73 oC. The slides were then hybridized in a moist chamber at 37 oC overnight.
Post hybridization slides were washed in 0.4XSSC/0.3% IGEPAL (Sigma) at 73 oC for 2 minutes and once in 2XSSC/0.1%IGEPAL at room temperature for one minute and counterstained with 4,6-diamidino phenylindole (DAPI) in antifade. A total of 600 cells were analyzed for individual patient using Zeiss Axioskop 2 plus microscope using triple bandpass filter.
Results
Of the total 105 CHD patients, Atrial septal defect (ASD) were 30 (28.57%), Ventricular Septal Defect (VSD) were 25 (23.80%), Teratology of Fallot (TOF) were 25 (23.80%), Coarctation of Aorta (COA) were 15 (14.28%) and TA (Truncus Arteriosus) were 10 (9.52%) table1. Clinically none of the patients showed any extra cardiac anomalies.
Cytogenetic analysis of all the 105 patients revealed a normal chromosomal complement. No additional chromosomal defects were observed in these patients. Parents of affected children were also subjected to cytogenetic analysis who revealed a normal karyotype.
FISH analysis on all the 105 patients revealed that only 6 (5.71%) out of them had the del22q11 table2 Figure1,Figure2. Of the 6 patients who showed the microdeletion, 3 had VSD (12%), 2 showed ASD (6.66%) and 1(4%) was a TOF patient table3. The parents of these 6 patients were further subjected to FISH analysis to know the possible origin of the microdeletion. FISH results revealed normal status of chromosome 22 in all the parents, thus confirming the de novo origin of deletions in patients.
Discussion
The present investigation was carried out to account for the microdeletion of 22q11 in isolated CHDs in Indian population. Karyotypic analysis did not reveal del22q11 in any of the 105 isolated CHD patients. FISH analysis using TUPLE1 probe on all the 105 isolated CHD patients involved in the study revealed microdeletion in 5.71% (6/105) of the cases with none of them showing any major or minor extra cardiac features. Studies by McDonald-McGinn et al., (1994)[21] and Amati et al ., (1995) [22] have suggested a correlation between 22q11.2 microdeletion and isolated heart defects with a detection rate of the deletion varying from 0% to 65% respectively. Contradicting to this, the studies carried out by Fokstuen et al (1998)[17] and Borgmann et al (1999)[18] with a sample size of 110 (syndromic + non-syndromic) and 175 (non-syndromic) respectively, the percentage of del22q11.2 was 0% in non-syndromic cases. In case of syndromic patients there was a considerable number of del22q11, which clearly indicates that microdeletion test should be performed in cases when extracardiac anomalies are present in addition to CHD. However, in the present investigation we find a small degree of association between isolated CHDs and 22q11 microdeletion which suggests that FISH analysis should be performed in non- syndromic CHD patients even when there are mild, unspecific extracardiac anomalies or no extracardiac anomalies. The origin of del22q11 is a debatable issue till date, as there are reports of both maternal and paternal involvement.[17] However, in the present study in all the 6 patients with del22q11the origin was de novo .
Present study is the third in series investigating the incidence of 22q11.2 deletions in patients with isolated CHDs. Although the sample size in our study was relatively small, further extensive studies are required to determine the exact frequency of deletions in Indian population. Due to the wide range and extremely variable expression of extracardiac anomalies, a careful general clinical examination of each patient with CHD is compulsory. We suggest that patients with VSD, ASD, TOF, COA, and TA with or without additional heart defects in combination with dysmorphic features (mild, severe or without) should be advised for FISH investigation for 22q11.2 deletion. Thus, with 22q11.2 microdeletion investigation in isolated CHD patients, an appropriate genetic counseling can be offered with accurate estimation of recurrence risk and possibility of prenatal diagnosis in further pregnancies.
Acknowledgements
This publication (NIRRH/MS/2/2005) has been supported by the Indian Council of Medical Research. The authors acknowledge Dr. C. P. Puri, Director, NIRRH, for the support given to this study.
References
1. Shi YR, Hsieh KS, Wu JY, Lee CC, Tsai CH, Tsai FJ. Molecular analysis of syndromic congenital heart diseases using short tandem repeat markers and semi quantitative polymerase chain reaction method. Pediatr Int 2002; 44 : 264-268.
2. Tezenas du Montal S, Mendizabal H, Ayme S, Levy A, Philip N. Prevalence of 22q11 microdeletion. J Med Genet 1996; 33: 719.
3. Devriendt K, Fryns J-P, Mortier G. The annual incidence of DiGeorge/Velocardiofacial syndrome. J Med Genet 1998; 35: 789-790.
4. Fernhoff PM: The 22q11.2 deletion syndrome: more answers but more questions. J Pediatrics 1998; 137: 145-147.
5. Payne M, Johnson MC, Grant JW and Strauss AW. Towards a molecular understanding of congenital heart disease. Circulation 1995; 91: 494-504.
6. http://www.americanheartassociation.com/Heart disease and stroke statistics-2003 update
7. Frias JL. Genetic issues of congenital heart disease. In: Gessner IH, Victrorica BE (eds) Pediatric Cardiology . Saunders, Philadelphia, 1993; 237-242.
8. Driscoll DA, Budarf ML, Emanuel BS. A genetic etiology for DiGeorge syndrome: consistent deletions and microdeletions of 22q11. Am J Hum Genet 1992; 50: 924-933.
9. Goldmuntz E, Driscoll D, Budarf ML, Zackai EH, McDonald McGinn DM, Biege JA, Emanuel BS. Microdeletions of chromosomal region 22q11 in patients with congenital conotruncal cardiac defects. J Med Genet 1993; 30: 807-812.
10. Burn J, Takao A, Wilson D, Cross I, Momma K, Wadey R, Scambler P, Goodship J. Conotruncal anomaly face syndrome is associated with a deletion within chromosome 22q11. J Med Genet 1993; 30: 822-824.
11. Giannotti A, Digilio MC, Marino B, Mingarelli R, Dallapicola B. Cayler cardiofacial syndrome and del22q11: part of the CATCH22 phenotype. Am J Med Genet 1994; 30 : 807-812.
12. Ryan AK, Goodship JA, Wilson DI, Philip N, Levy A, Seidel H et al. Spectrum of clinical features associated with interstitial chromosome 22q1 deletions: A European collaborative study. J Med Genet 1997; 34: 798-804.
13. Wilson DI, Burn J, Scambler P, Goodship J. DiGeorge syndrome: part of CATCH 22. J Med Genet 1993; 30: 852-856.
14. Demezuk S, Aurias A. DiGeorge syndrome and related syndromes associated with 2q11.2 deletions-a review. Ann Genet 1995; 38; 59-76.
15. Wilson DI, Goodship JA, Scambler PJ, Carey A, Cross I, Burn J. Is monosomy for the DiGeorge locus on chromosome 22 responsible for isolated heart malformations Am J Hum Genet 1991; 49 [Suppl]: 901.
16. Wilson DI, Goodship JA, Burn J, Cross IE, Scambler PJ. Deletions within chromosome 22q11 in familial congenital heart disease. Lancet 1992; 340 : 573-575.
17. Fokstuen S, Arbenz U, Artan S, Dutly F, Bauersfeld U, Brecevic L, Fasnacht M, Rothlisberger B, Schizel A. 22q11.2 deletions in a series of patients with non-selective congenital heart defects: incidence, type of defects and parental origin. Clin Genet 1998; 53: 63-69.
18. Borgmann S, Luhmer I, Arslan-Kirchner M, Kallfelz HC, Schmidtke J. A search for chromosome 22q11.2 deletions in a series of 176 consecutively catheterized patients: no evidence for deletions in non-syndromic patients. Eur J Pedia 1999; 158: 958-963.
19. Seabright M. A rapid banding technique for human chromosomes. Lancet 1971: 2 : 971-972.
20. International System for Human Cytogenetic Nomenclature (ISCN 1995) Editor: Felix Mitelman, Karger publication.
21. McDonald-McGinn DM, Driscoll DA, Goldmuntz E, Clark BJ, LaRossa D, Randall P, Cohen M, Solat C, Schultz P, Zackai EH. Velopharyngeal incompetence diagnosed in a series of cardiac patients prompted by the findings of a 22q11.2 deletion. Am J Hum Genet 1994; 55: 35.
22. Amati F, Mari A, Digilio MC, Mingarelli R, Marino B, Giannoti A, Novelli G, Dallapiccola B. 22q11 deletions in isolated and syndromic patients with tetralogy of fallot. Hum Genet 1995; 95: 479-482.(Gawde H, Patel ZM, Khatkh)
2 Department of Cardiology, Cardiovascular Thoracic Center, King Edward Memorial (KEM) Hospital, Parel, Mumbai, India
Abstract
Objective. To analyze the frequency of del22q11.2 in non-syndromic CHDs using classical cytogenetics and Fluorescence In Situ Hybridization (FISH) technique in Indian population. Methods. 105 prospective cases which included 6 families with isolated, non-syndromic cardiac defects were analyzed clinically by a cardiologist and a geneticist. The cases were then subjected to karyotypic (classical cytogenetics) as well as FISH analysis. The efficacy of FISH technique was compared with inference drawn from classical cytogenetics. Results. Karyotypic analysis of all the 105 patients revealed a normal chromosomal complement. Microdeletion 22q11.2 was observed in six patients (5.71%) by FISH studies. FISH studies were also performed on the parents of these six patients who revealed a normal chromosome 22. No correlation was found between clinical features (mild or unspecific) with 22q11.2 microdeletion. Conclusion. The testing for microdeletion 22q11.2 in isolated non-syndromic patients using FISH technique is mandatory even when mild/unspecific extracardiac abnormalities are seen in the patients.
Keywords: FISH; India; Isolated congenital heart disease (CHD); Microdeletion 22q11.2
Congenital heart diseases (CHDs) are the most common of all birth defects and are the leading cause of mortality in the first year of life with a prevalence of 1% in live births and 10% in spontaneously aborted fetus.[1] One of the most common syndromes associated with CHDs is the 22q11.2 deletion (del22q11.2) syndrome, which has an estimated prevalence of approximately 1 in 4000 live births [2], [3] of which 90% of cases are sporadic.[4] CHDs are a multifactorial defects caused by (a) 90% multifactorial disorders (b) chromosomal disorders and single gene disorders constituting 8% and (c) 2% of environmental teratogens.[5] They are the most common single group of congenital abnormalities accounting for about 30% of the total abnormalities.[6] In most patients, CHDs occur as an isolated malformation, but about 33% have associated anomalies [7].
The various conditions associated with del22q11 include DiGeorge anomaly (DG), Velocardiofacial syndrome (VCFS), Conotruncal anomaly face syndrome (CTAFS), isolated conotruncal heart defects (CTHDs) and Cayler cardiofacial syndrome.[8],[9],[10],[11] The abnormalities that are already known to be associated with this deletion affect multiple body systems, including cranofacial and otolaryngeal structures, cardiovascular, endocrine, nervous, musculoskeletal and immune processes.[12] As a result of their common genetic origin and their phenotypic overlap the syndromes were collectively referred to as "CATCH 22" (Cardiac abnormality, Abnormal facies, Thymic hypoplasia, Cleft palate, Hypocalcemia and del22q11.2.[11],[13] The cardiac defects commonly seen in del22q11.2 syndrome are derived from conotruncus, the embryonic aortic arches and the ventricular septum which are Tetralogy of Fallot (TOF), Ventricular Septal Defect (VSD), Atrial Septal Defect (ASD), Truncus Arteriosus (TA), and Interrupted Aortic Arch (IAA). [14]
Previous studies on the presence of 22q11.2 microdeletion in patients with isolated CHDs have been reported since the early 1990s.[9],[15],[16] However, such studies have not been taken up in India till date. The present investigation was initiated in order to look into the issue of microdeletion 22q11.2 in a non-selected patient group in Indian population and to develop genetic guidelines for microdeletion testing which will help in future evaluation of patients with conotruncal defects. Two similar studies in unselected patients with CHD have been reported [17],[18] and ours is the third in the series.
Materials and methods
In the present study, a total of 105 subjects (consecutive, non-selected) were enrolled prospectively from OPD of Cardio Vascular Thoracic Center (CVTC), KEM hospital as well as from Genetic Research Center Clinic (NIRRH). The subjects belonged to the age group ranging from 24 days to 24 years. The cardiologist from CVTC clinically confirmed the CHD by ECG, 2D Echocardiography and Colour Doppler study in all subjects included in the present investigation. The clinical geneticist ascertained the clinical data, which included weight, height, type of CHD, dysmorphisms, extra cardiac malformations (if any) and psychomotor development.
Peripheral venous blood was obtained from each patient for karyotypic and Fluorescence in Situ Hybridization (FISH) analysis, with the consent of the family members. Chromosomal analysis was carried out on peripheral blood lymphocyte culture using standard protocol with suitable modifications (Seabright 1971). [19] G-banded metaphase chromosomes were screened at 550-band level. The chromosomes were analyzed and karyotyped according to the International System for Human Cytogenetic Nomenclature (ISCN 1995). [20]
Fluorescence in situ Hybridization (FISH) was performed on metaphase chromosome spreads using the VYSIS LSI DiGeorge syndrome probe which maps to the TUPLE1 region i.e. 22q11.2 (spectrum orange) combined with 22q11.3 (spectrum green) region control probe according to manufacturers instructions.
In short, slides were treated with 70% acetic acid for 15 minutes at room temperature, then dehydrated in a series of alcohol grades 70%, 85% 100% for 3 minutes each, then treated with pepsin (1mg/ml) final stock for 3 minutes at 37 oC. The slides were then washed in 1XPBS for 5 minutes, dehydrated in a series of alcohol grades for 3 minutes each and air-dried. Probe mixture was prepared according to manufacturers instructions. The probe mixture was then applied to the slide, coverslip placed, sealed with rubber cement and slide was subjected to co-denaturation on a hot plate for 4 minutes at 73 oC. The slides were then hybridized in a moist chamber at 37 oC overnight.
Post hybridization slides were washed in 0.4XSSC/0.3% IGEPAL (Sigma) at 73 oC for 2 minutes and once in 2XSSC/0.1%IGEPAL at room temperature for one minute and counterstained with 4,6-diamidino phenylindole (DAPI) in antifade. A total of 600 cells were analyzed for individual patient using Zeiss Axioskop 2 plus microscope using triple bandpass filter.
Results
Of the total 105 CHD patients, Atrial septal defect (ASD) were 30 (28.57%), Ventricular Septal Defect (VSD) were 25 (23.80%), Teratology of Fallot (TOF) were 25 (23.80%), Coarctation of Aorta (COA) were 15 (14.28%) and TA (Truncus Arteriosus) were 10 (9.52%) table1. Clinically none of the patients showed any extra cardiac anomalies.
Cytogenetic analysis of all the 105 patients revealed a normal chromosomal complement. No additional chromosomal defects were observed in these patients. Parents of affected children were also subjected to cytogenetic analysis who revealed a normal karyotype.
FISH analysis on all the 105 patients revealed that only 6 (5.71%) out of them had the del22q11 table2 Figure1,Figure2. Of the 6 patients who showed the microdeletion, 3 had VSD (12%), 2 showed ASD (6.66%) and 1(4%) was a TOF patient table3. The parents of these 6 patients were further subjected to FISH analysis to know the possible origin of the microdeletion. FISH results revealed normal status of chromosome 22 in all the parents, thus confirming the de novo origin of deletions in patients.
Discussion
The present investigation was carried out to account for the microdeletion of 22q11 in isolated CHDs in Indian population. Karyotypic analysis did not reveal del22q11 in any of the 105 isolated CHD patients. FISH analysis using TUPLE1 probe on all the 105 isolated CHD patients involved in the study revealed microdeletion in 5.71% (6/105) of the cases with none of them showing any major or minor extra cardiac features. Studies by McDonald-McGinn et al., (1994)[21] and Amati et al ., (1995) [22] have suggested a correlation between 22q11.2 microdeletion and isolated heart defects with a detection rate of the deletion varying from 0% to 65% respectively. Contradicting to this, the studies carried out by Fokstuen et al (1998)[17] and Borgmann et al (1999)[18] with a sample size of 110 (syndromic + non-syndromic) and 175 (non-syndromic) respectively, the percentage of del22q11.2 was 0% in non-syndromic cases. In case of syndromic patients there was a considerable number of del22q11, which clearly indicates that microdeletion test should be performed in cases when extracardiac anomalies are present in addition to CHD. However, in the present investigation we find a small degree of association between isolated CHDs and 22q11 microdeletion which suggests that FISH analysis should be performed in non- syndromic CHD patients even when there are mild, unspecific extracardiac anomalies or no extracardiac anomalies. The origin of del22q11 is a debatable issue till date, as there are reports of both maternal and paternal involvement.[17] However, in the present study in all the 6 patients with del22q11the origin was de novo .
Present study is the third in series investigating the incidence of 22q11.2 deletions in patients with isolated CHDs. Although the sample size in our study was relatively small, further extensive studies are required to determine the exact frequency of deletions in Indian population. Due to the wide range and extremely variable expression of extracardiac anomalies, a careful general clinical examination of each patient with CHD is compulsory. We suggest that patients with VSD, ASD, TOF, COA, and TA with or without additional heart defects in combination with dysmorphic features (mild, severe or without) should be advised for FISH investigation for 22q11.2 deletion. Thus, with 22q11.2 microdeletion investigation in isolated CHD patients, an appropriate genetic counseling can be offered with accurate estimation of recurrence risk and possibility of prenatal diagnosis in further pregnancies.
Acknowledgements
This publication (NIRRH/MS/2/2005) has been supported by the Indian Council of Medical Research. The authors acknowledge Dr. C. P. Puri, Director, NIRRH, for the support given to this study.
References
1. Shi YR, Hsieh KS, Wu JY, Lee CC, Tsai CH, Tsai FJ. Molecular analysis of syndromic congenital heart diseases using short tandem repeat markers and semi quantitative polymerase chain reaction method. Pediatr Int 2002; 44 : 264-268.
2. Tezenas du Montal S, Mendizabal H, Ayme S, Levy A, Philip N. Prevalence of 22q11 microdeletion. J Med Genet 1996; 33: 719.
3. Devriendt K, Fryns J-P, Mortier G. The annual incidence of DiGeorge/Velocardiofacial syndrome. J Med Genet 1998; 35: 789-790.
4. Fernhoff PM: The 22q11.2 deletion syndrome: more answers but more questions. J Pediatrics 1998; 137: 145-147.
5. Payne M, Johnson MC, Grant JW and Strauss AW. Towards a molecular understanding of congenital heart disease. Circulation 1995; 91: 494-504.
6. http://www.americanheartassociation.com/Heart disease and stroke statistics-2003 update
7. Frias JL. Genetic issues of congenital heart disease. In: Gessner IH, Victrorica BE (eds) Pediatric Cardiology . Saunders, Philadelphia, 1993; 237-242.
8. Driscoll DA, Budarf ML, Emanuel BS. A genetic etiology for DiGeorge syndrome: consistent deletions and microdeletions of 22q11. Am J Hum Genet 1992; 50: 924-933.
9. Goldmuntz E, Driscoll D, Budarf ML, Zackai EH, McDonald McGinn DM, Biege JA, Emanuel BS. Microdeletions of chromosomal region 22q11 in patients with congenital conotruncal cardiac defects. J Med Genet 1993; 30: 807-812.
10. Burn J, Takao A, Wilson D, Cross I, Momma K, Wadey R, Scambler P, Goodship J. Conotruncal anomaly face syndrome is associated with a deletion within chromosome 22q11. J Med Genet 1993; 30: 822-824.
11. Giannotti A, Digilio MC, Marino B, Mingarelli R, Dallapicola B. Cayler cardiofacial syndrome and del22q11: part of the CATCH22 phenotype. Am J Med Genet 1994; 30 : 807-812.
12. Ryan AK, Goodship JA, Wilson DI, Philip N, Levy A, Seidel H et al. Spectrum of clinical features associated with interstitial chromosome 22q1 deletions: A European collaborative study. J Med Genet 1997; 34: 798-804.
13. Wilson DI, Burn J, Scambler P, Goodship J. DiGeorge syndrome: part of CATCH 22. J Med Genet 1993; 30: 852-856.
14. Demezuk S, Aurias A. DiGeorge syndrome and related syndromes associated with 2q11.2 deletions-a review. Ann Genet 1995; 38; 59-76.
15. Wilson DI, Goodship JA, Scambler PJ, Carey A, Cross I, Burn J. Is monosomy for the DiGeorge locus on chromosome 22 responsible for isolated heart malformations Am J Hum Genet 1991; 49 [Suppl]: 901.
16. Wilson DI, Goodship JA, Burn J, Cross IE, Scambler PJ. Deletions within chromosome 22q11 in familial congenital heart disease. Lancet 1992; 340 : 573-575.
17. Fokstuen S, Arbenz U, Artan S, Dutly F, Bauersfeld U, Brecevic L, Fasnacht M, Rothlisberger B, Schizel A. 22q11.2 deletions in a series of patients with non-selective congenital heart defects: incidence, type of defects and parental origin. Clin Genet 1998; 53: 63-69.
18. Borgmann S, Luhmer I, Arslan-Kirchner M, Kallfelz HC, Schmidtke J. A search for chromosome 22q11.2 deletions in a series of 176 consecutively catheterized patients: no evidence for deletions in non-syndromic patients. Eur J Pedia 1999; 158: 958-963.
19. Seabright M. A rapid banding technique for human chromosomes. Lancet 1971: 2 : 971-972.
20. International System for Human Cytogenetic Nomenclature (ISCN 1995) Editor: Felix Mitelman, Karger publication.
21. McDonald-McGinn DM, Driscoll DA, Goldmuntz E, Clark BJ, LaRossa D, Randall P, Cohen M, Solat C, Schultz P, Zackai EH. Velopharyngeal incompetence diagnosed in a series of cardiac patients prompted by the findings of a 22q11.2 deletion. Am J Hum Genet 1994; 55: 35.
22. Amati F, Mari A, Digilio MC, Mingarelli R, Marino B, Giannoti A, Novelli G, Dallapiccola B. 22q11 deletions in isolated and syndromic patients with tetralogy of fallot. Hum Genet 1995; 95: 479-482.(Gawde H, Patel ZM, Khatkh)