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Prevalence and Parental Origin in Tetralogy of Fallot Associated With Chromosome 22q11 Microdeletion

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Prevalence and Parental Origin in Tetralogy of Fallot Associated With

Chromosome 22q11 Microdeletion

Jen-Her Lu, MD, PhD*; Ming-Yi Chung, PhD‡; Betau Hwang, MD*; and Hsieh-Ping Chien, MS*

ABSTRACT. Objective. Tetralogy of Fallot is a com-mon cardiac anomaly that is associated with chromosome 22q11 microdeletion. In this study we examined the mode of transmission as well as the parental origin of microdeletion in patients with tetralogy of Fallot.

Methods. Eighty-four children with sporadic tetral-ogy of Fallot (40 boys and 44 girls; mean age, 34 months) were analyzed for microdeletion at chromosome 22q11 by genotype analysis, using five microsatellite markers, D22S427, D22S941, D22S944, D22S264 and D22S311, and confirmed by quantitative polymerase chain reaction, us-ing TUPLE1 and D22S264. All parents of these subjects consented to their own participation and their child’s participation in the clinical evaluation and molecular study. To provide a molecular characterization of mi-crodeletion, we isolated DNA from the parents and typed their DNA with each of the five polymorphic markers.

Results. Sixty-six patients were associated with pul-monary stenosis; and 8 of these cases (12%) had microde-letion. Eighteen patients were associated with pulmonary atresia, and 6 (33%) of these cases had microdeletion. The parental origins of the 14 patients with microdeletion were paternal in 3 cases and maternal in 11 cases. The most common mode of transmission was de novo with-out parental hemizygosity (93%). Transmission by auto-somal dominant heredity was uncommon (7%).

Conclusions. Biased parental origin was consistently found in tetralogy of Fallot patients with chromosomal 22q11 microdeletion. Our results indicated a higher prev-alence of microdeletion because of inheritance of mater-nal microdeletion (78%).Pediatrics1999;104:87–90; tetral-ogy of Fallot, CATCH 22, conotruncal malformation DiGeorge/velocardiofacial syndrome.

ABBREVIATIONS. del22q11, microdeletion of chromosome 22q11; DG/VCF, DiGeorge/velocardiofacial; TF, tetralogy of Fal-lot; PCR, polymerase chain reaction; PS, pulmonary stenosis; PA, pulmonary atresia.

T

he chromosome 22q11 microdeletion (del22q11) is a well-established genetic origin of DiGeorge syndrome, conotruncal facial syndrome, and ve-locardiofacial syndrome.1–3 These syndromes have

been incorporated into a group under the acronym CATCH 22 (Conotruncal cardiac defect, Abnormal face, Thymic hypoplasia,Cleft palate, Hypocalcemia,

microdeletion 22q11).4,5Because of the negative

conno-tations of the term CATCH 22, it has been proposed that the compound be called DiGeorge/velocardiofa-cial (DG/VCF) syndrome to emphasize its broad phe-notypic spectrum.6A large documented proportion of

these patients are hemizygous for part of 22q11, sug-gesting that haploinsufficiency of a gene(s) within chromosome 22q11 is responsible for the DG/VCF syndromes.7–9More recently, del22q11 has been

docu-mented among patients with conotruncal heart de-fects.8 Besides interrupted aortic arch, tetralogy of

Fallot (TF) is the most common type of cardiac anomaly seen with del22q11.10,11In this study, we examined the

mode of transmission as well as the parental origin of the del22q11 in patients with TF by constructing a physical map containing a set of five ordered simple tandem repeat polymorphic markers. These markers were used to define the deletion in each patient by detecting the loss of heterozygosity of a particular marker when compared with the parents.

METHODS

Eighty-four consecutive patients with TF (40 males and 44 females; mean age, 34 months) were evaluated during the period from February 1996 to March 1997. Diagnosis of TF was deter-mined by echocardiographic evidence of overriding aorta or an-terior deviation of the coronal septum with ventricular septal defect. Echocardiographic findings were confirmed by cardiac catheterization and/or cardiac surgery in all patients. The clinical spectrum for each patient was evaluated for parameters including growth percentile, cardiac defect, dysmorphic face, palatal abnor-malities, T-cell counts (CD3/CD4/CD8), serum ionized calcium, neuromuscular development, and learning disability. Phytohe-magglutinin antigen (PHA)-stimulated T-cell proliferation was also studied in patients with del22q11. Each patient was evaluated for dysmorphic features and the presence of thymus at the time of cardiac surgery. The clinical diagnosis of DG/VCF syndrome was mainly made based on typical facial appearance, the appearance of dysmorphic features, neonatal hypocalcemia, and thymic hy-poplasia.

Chromosomal Analysis and DNA Extraction

Blood samples from all TF patients and their parents were collected in an institutional review board-approved program, and DNA was prepared (Puregene DNA kit; Gentra, Minneapolis, MN). Standard chromosomal analyses were performed in all patients.

Simple Tandem Repeat Polymorphic Marker Analysis

Microdeletion of chromosome 22q11 was determined by geno-type analysis using five microsatellite markers. Their relative po-sition on 22q is cen-D22S427-D22S941-D22S944-D22S264-D22S311-qter (Fig 1). Polymerase chain reaction (PCR) amplification was conducted in 10-mL reactions for each locus with 20 ng of genomic DNA, 200 mmol/L dNTPs, 0.25 U of AmpliTaq Gold (Perkin-Elmer, Branchburg, NJ), and 13reaction buffer supplied with the enzyme (10 mmol/L Tris, pH 8.3, 1.5 mmol/L KCl, and 0.001%

From the Departments of *Pediatrics and ‡Pathology and Laboratory Med-icine, Veterans General Hospital-Taipei, National Yang-Ming University, Taipei, Taiwan, Republic of China.

Received for publication Aug 20, 1998; accepted Nov 23, 1999.

Reprint requests to (J.-H.L.) Department of Pediatrics, Veterans General Hospital-Taipei, Taipei, Taiwan, ROC.

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gelatin). Each reaction went through an initial denaturation at 94°C for 7 minutes, followed by 30 cycles of 94°C for 1 minute, 55°C for 1 minute, and 72°C for 1 minute. Stop solution (7ml) was then added to the PCR product, which was then denatured at 95°C for 5 minutes and resolved on a 4% denaturing polyacrylamide gel containing 8 mmol/L urea. After electrophoresis, DNA on the gel was transferred onto a Tropilon-plus nylon membrane (Tropix, Bedford, MA) and detected by chemiluminescence (Tropix).

Quantitative Multiplex PCR

Quantitative multiplex PCR was performed for two chromo-some 22q11 loci, TUPLE1 and D22S264, with D10S2198 and b-glo-bin chain as an internal control, respectively (Fig 2). The positions of these TUPLE1 and D22S264 probes within the physical map of 22q11 are shown in Fig 1. Twenty nanograms of genomic DNA was used to set up a 20-mL PCR reaction with 10mmol/L of each primer, 200 mmol/L of dNTPs, and 0.5 U of AmpliTaq Gold (Perkin-Elmer) and 13reaction buffer supplied with the enzyme (10 mmol/L Tris, pH 8.3, 1.5 mmol/L MgCl2, 50 mmol/L KCl, and 0.001% gelatin). The reaction was conducted with an initial dena-turation at 94°C for 7 minutes, followed by 25 cycles of 94°C for 1 minute, 55°C for 1 minute, and 72°C for 1 minute. Four microliters of loading dye was added to the 20-mL multiplex reaction. Fifteen microliters of the mixture was loaded onto a 2% agarose gel with 2mL of Low DNA Mass Ladder (GibcoBRL, Grand Island, NY) and run at 150 V for'1.5 hours. After electrophoresis, the gel was stained for 15 minutes in solution containing 0.5mg/mL ethidium bromide. The image was then captured before saturation under the following conditions: Black was set at 0, white at 255, contrast value,g, at 1.0, and aperture at 28, using a cooled-charge coupled device camera. The captured file was saved as a TIFF bitmap file for quantitative analysis, which was performed on a Macintosh computer, using the public domain National Institutes of Health Image program (Version 1.61; developed at the US National Insti-tutes of Health and available on the Internet at http://rsb.inf. nih.gov/nih-image).

Parental Origin of the Microdeletion

All parents of the patients with microdeletion (n514) gave informed consent to participate in the clinical evaluation and molecular study. To provide a molecular characterization of del22q11 in TF, we isolated DNA from all parents and typed their DNA with each of the five polymorphic markers (Table 1). Rep-resentative results of this analysis are illustrated in Fig 3.

Using Quantitative Multiplex PCR to Confirm the Microdeletion

Quantitative multiplex PCR for TUPLE1 and D22S264 was conducted to confirm the results of genotyping. The parental origin of the del22q11 and whether each case was a new mutation or inherited from one of the parents were established by

quanti-tative multiplex PCR analysis, using samples from parents of the subjects with the deletion. For each family with a deleted case, the observation of heterozygous status at the loci where the deletion had occurred in the parent from whom the deleted chromosome homolog was derived was taken as indication of a de novo mu-tation. In hereditary cases, the homozygous/hemizygous status throughout several loci in both the parent and his/her offspring was determined.

Statistics

x2analysis of parental origin was used for group comparisons, and a value ofP,.05 was considered statistically significant.

RESULTS

A clinical diagnosis of DG/VCF syndrome was made in 27 cases. Other syndromes diagnosed in-cluded Down syndrome in 5 cases and CHARGE (Coloboma, Heart disease, Atresia choanae, Re-tarded growth and reRe-tarded development and/or central nervous system anomalies,Genital hypopla-sia, and Ear anomalies and/or deafness) syndrome in 2 cases. The del22q11 was identified in 14 of the 27 patients with DG/VCF syndrome. Dysmorphic face (n 5 14; 100%), learning disability (n 5 14; 100%), cleft palate (n 5 6, 42%), and delayed neurological development (n5 13;.95%) were very common in these patients. There were no statistically significant differences in the clinical findings between DG/VCF

Fig 1. Map of the DiGeorge critical region on chromosome 22q11.2. Localization of polymerase chain reaction microsatelites markers and the TUPLE1 gene used in this study are labeled.

Fig 2. Representative example of microdeletion from maternal (A) and paternal origins (B) determined by polymerase chain reaction microsattelite analysis at D22S427, D22S944, D22S264, and D22S311. Alleles marked by double arrowheads (..and,,) in the affected individuals of A (patient 11) and B (patient 27) showed failure to inherit paternal and maternal alleles, respec-tively.

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patients with (n514) or without (n513) del22q11. The case with the smallest deletion (detected only at D22S427) had the highest growth percentile at the age of 2 years. All the other cases with del22q11 showed relatively poor growth, usually within the 25th percentile, and as low as the 5th percentile (.70%). Thymic function, evaluated by T-cell counts (CD3/CD4/CD8), was within the reference ranges in all cases with del22q11. Phytohemagglutinin antigen (PHA)-stimulated T-cell proliferation was mostly normal (75%; 9 of 14 cases).

Sixty-six (79%) of the patients with TF had the classic anatomic form with pulmonary stenosis (PS), and 18 had pulmonary atresia (PA). The chromo-some 22q11 microdeletion was found in 33% (6 of 18) of the children with PA, and 12% (8 of 66) of the children with PS had del22q11 (Fig 4). The parental origin of the deleted chromosomes was determined, with 3 of paternal and 11 of maternal origin. Most of

the patients with del22q11 (13 of 14 cases; 93%) had a de novo deletion without parental hemizygosity. Eleven cases with del22q11 were born to mothers younger than 35 years old (80%). Inherited cases were relatively rare, with only 1 in 14 cases (7%). The mother of this patient was phenotypically character-istic, presenting with conotruncal facial syndrome with 22q11 hemizygosity. The microdeletion was maternally derived in 5 of the 6 TF patients with PA (83%). Among the del22q11 in 8 TF patients with PS, 6 originated maternally and 2 paternally (75%). The higher prevalence of maternal origin in these two types of TF with deleted chromosomes was statisti-cally significant (P,.05).

DISCUSSION

Heterogeneity of phenotype and various patterns of inheritance have been observed in association with DG/VCF syndrome, including sporadic, auto-somal dominant, and autoauto-somal recessive. To date, no molecular difference has been reported that can explain the phenotypic variability of DG/VCF syn-drome.6 – 8Genetic backgrounds such as unstable

mu-tations, modified genes, the presence of an imprinted locus in this region, or parent-of-origin effects may ultimately explain the variability in clinical manifes-TABLE 1. Genotype Analysis of TF Patients With Microdeletion

Patient No.

PCR Markers Parental

Origin D22

S427

D22 S941

D22 S944

D22 S264

D22 S311

1 UI D D D D Mat

2 D UI UI No No Pat

3 No D D D UI Mat

4 UI D D D D Mat

5 UI D D D UI Mat

6 UI D D D UI Mat

7 No D D D UI Mat

8 No D D D UI Mat

9 No D D D D Pat

10 No D D D UI Mat

11 No D D D UI Mat

12 No D D D UI Mat

13 No D D D UI Pat

14 No D D D UI Mat

Abbreviations: TF, tetralogy of Fallot; PCR, polymerase chain reaction; D, deletion; No, no deletion; UI, uninformative; Mat, maternal; Pat, paternal.

Fig 3. Quantitative multiplex polymerase chain reaction (PCR) at TUPLE1 and D22S264 (A) and densitometry analysis of the gel for quantitative multiplex PCR (B). Case 1 (lanes 1) is a control with-out del22q11; case 2 (lanes 2) is patient 6 with del22q11. Loci tested (TUPLE1 and D22S264) and their internal controls (D10S2198 and HB) are marked at the top for the gel and at the left side for the densitometric analysis.

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tations. Polymorphic short tandem repeats are cur-rently being used to provide parent-of-origin infor-mation for at-risk patients. The identification of molecular genetic alterations in association with conotruncal cardiac defects enables accurate genetic counseling to be given to the family. Identification of a de novo deletion suggests that the parents of an affected child have a negligible risk of recurrence.

In this study, we found that the most common mode of transmission was de novo without parental hemizygosity. Based on the 3% risk of recurrence among offspring of individuals affected with TF, Wilson et al5suggested that, if all the recurrent cases

resulted from del22q11,'6% of the affected individ-uals would be expected to carry this chromosomal abnormality. The reported prevalence of del22q11 in TF has ranged from 8% to 16%.2– 6Debrus et al3found

that familial nonsyndromic conotruncal defects are not associated with a 22q11 microdeletion. This in-dicates that a significant number of new mutations do occur in addition to the estimated 6% among inherited cases. Furthermore, the underlying causes of microdeletion can be demonstrated to be the result of a low incidence (1.2%) of inheritance and a high incidence (14.8%) of de novo mutation. The low in-cidence of inherited del22q11 implies that the trans-mission of the deletion to the next generation is impaired, although infertility of syndromic TF has not been documented. The high incidence of de novo mutation suggests that new mutations are actually the main cause of TF with del22q11.

In theory, the del22q11 is a mutation of autosomal chromosomes that is transmitted to offspring at a rate of 50% without parental difference. Several re-ports have suggested that there is not a consistent parent of origin or inherited unbalanced transloca-tion in DiGeorge patients.6 – 8 A preponderance of

maternally derived deletions in TF patients with PA has been suggested previously.10Our results suggest

that there is a biased parental origin for the del22q11 in all children with TF. Although the sample sizes of each category remained relatively small, biased pa-rental transmission was found in both patients with TF-PA (83%) and patients with TF-PS (75%). In this study, most of the TF patients with del22q11 derived these mutations from their mothers. The reason for the preferential occurrence of parental deletion in sporadic congenital heart disease remains unclear. The lack of documentation of infertility in males affected with DG/VCF syndrome fails to support the notion that fertility is decreased among these males. However, the preferential maternal origin could be the result of decreased reproductive success or de-creased fertility in males. The higher frequency of deletions of a maternal origin could be attributed to several causes, including imprinting of the chromo-somal region, greater susceptibility to deletion of the maternal chromosome, or an unexplained preferen-tial rearrangement of maternal chromosome 22. However, no mapping of imprinted genes to chro-mosome 22 in the DiGeorge critical region has been reported.

Early identification of TF patients with DG/VCF syndrome facilitates the diagnosis and management of associated manifestations such as immune disor-ders, growth delay, learning disabilities, speech dis-turbances, renal anomalies, brochospasms, psychiat-ric disorders, and ocular disorders. Amati et al4

reported that none of the patients with isolated TF had del22q11. We found also in this study that TF patients with del22q11 always presented some extra-cardiac anomalies, including most frequently with subtle facial dysmorphisms, and mental and/or growth retardation. However, the phenotypes of DG/VCF syndrome in TF patients were very poly-morphic and anomalies were sometimes very subtle, which rendered the clinical diagnosis difficult. Our results revealed that cytologically detectable hemizy-gous deletions of chromosome 22q11.2 were rela-tively frequent in all children with TF and more frequent in those with PA. In most patients, the del22q11 occurred in the DiGeorge critical region, with the microdeletion spanning 1 to 2 megabase in size, and was not visible in routine chromosome preparations. It is therefore relevant to suggest that determination of the presence or absence of del22q11 in all children with TF should be performed with either molecular analysis and/or high-quality (pro)-metaphase cytogenic preparations.

ACKNOWLEDGMENTS

This work was supported by the National Scientific Council, Taiwan, ROC (NSC 88 –2314-V-075– 036), and a grant from Veter-ans General Hospital-Taipei (V-337– 86).

REFERENCES

1. Wilson DI, Burn J, Scambler P, Goodship J. DiGeorge syndrome: part of CATCH22.J Med Genet.1993;30:852– 856

2. Momma K, Kondo C, Ando M, Matsuoka R, Takao A. Tetralogy of Fallot associated with chromosome 22q11 deletion.Am J Cardiol.1995; 76:618 – 621

3. Debrus S, Berger G, De Meeus A, Sauer U, Guillaumont S, Voisin M, et al. Familial non-syndromic conotruncal defects are not associated with a 22q11 microdeletion.Hum Genet.1996;97:138 –144

4. Amati F, Mari A, Digilio MC, Mingarelli R, Marino B, Giannotti A, et al. 22q11 deletions in isolated and syndromic patients with tetralogy of Fallot.Hum Genet.1995;95:479 – 482

5. Wilson DI, Goodship JA, Burn J, Cross IE, Scambler PJ. Deletions within chromosome 22q11 in familial congenital heart disease.Lancet.1995;340: 573–575

6. Morrow B, Goldberg R, Carlson C, Gupta DR, Sirotkin H, Collins J, et al. Molecular definition of the 22q11 deletions in velo-cardio-facial syn-drome.Am J Hum Genet.1995;56:1391–1403

7. Wulfsberg EA, Leana-Cox J, Neri G. What’s in a name? Chromosome 22q11 abnormalities and the DiGeorge, velocardiofacial, and conotrun-cal anomalies face syndromes.Am J Med Genet.1996;65:317–319 8. Carlson C, Papolos D, Pandita RK, Faedda GL, Veit S, Goldberg R, et al.

Molecular analysis of velo-cardio-facial syndrome patients with psychi-atric disorders.Am J Hum Genet.1997;60:851– 859

9. Goldmuntz E, Driscoll D, Budarf ML, Zackai EH, MacDonald-McGinn DM, Biegel JA, Emanuel BS. Microdeletions of chromosomal region 22q11 in patients with congenital conotruncal cardiac defects.J Med Genet.1993;30:807– 812

10. Digilio MC, Marino B, Grazioli S, Agostino D, Giannotti A, Dallapiccola B. Comparison of occurrence of genetic syndromes in ventricular septal defect with pulmonic stenosis versus ventricular septal defect with pulmonic atresia.Am J Cardiol.1996;78:1375–1376

11. Seaver LH, Pierpont JW, Erikson RP, Donnerstein RL, Cassidy SB. Pulmonary atresia associated with maternal 22q11.2 deletion: possible parent of origin effect in the conotruncal anomaly face syndrome.J Med Genet.1994;31:830 – 834

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DOI: 10.1542/peds.104.1.87

1999;104;87

Pediatrics

Jen-Her Lu, Ming-Yi Chung, Betau Hwang and Hsieh-Ping Chien

Chromosome 22q11 Microdeletion

Prevalence and Parental Origin in Tetralogy of Fallot Associated With

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DOI: 10.1542/peds.104.1.87

1999;104;87

Pediatrics

Jen-Her Lu, Ming-Yi Chung, Betau Hwang and Hsieh-Ping Chien

Chromosome 22q11 Microdeletion

Prevalence and Parental Origin in Tetralogy of Fallot Associated With

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Figure

Fig 1. Map of the DiGeorge critical region on chromosome22q11.2. Localization of polymerase chain reaction microsatelitesmarkers and the TUPLE1 gene used in this study are labeled.
Fig 4. Prevalence and parental origin of del22q11 in patients withtetralogy of Fallot (TF)

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