Interstitial deletion 13q31 associated with normal phenotype: cytogenetic study of a family with concomitant segregation of reciprocal translocation and interstitial deletion.

Gain or loss of a fragment in human chromosomes has been associated with abnormal phenotypes in numerous genetic disorders. However, it is also possible that lack or excess of a particular chromosomal segment is a neutral polymorphism among populations and thus does not cause obvious abnormal phenotype. In this study, conventional GTG-banded karyotyping and molecular cytogenetic analyses (including fluorescence in situ hybridization, spectral karyotyping and comparative genomic hybridization) were applied to study the genotype-phenotype correlation in a Taiwanese family, in which a concomitant segregation of del(13)(q31q31) interstitial deletion and t(13;18)(q32;p11.2) reciprocal translocation in a 2-year-old girl (the proband) was noticed. Two family members (the father and grandmother of the proband) who carried the del(13)(q31q31) but not the translocation t(13;18) both revealed a normal phenotype at adulthood. The finding, which appears novel, that interstitial deletion 13q31 could be associated with a normal phenotype, is therefore valuable in genetic counseling.

Persistent fifth aortic arch associated with 22q11.2 deletion syndrome.

BACKGROUND: Chromosome 22q11.2 deletion is frequently associated with conotruncal malformations and aortic arch anomalies. This study investigated the association of chromosome 22q11.2 deletion with clinical manifestations in four pediatric patients with persistent fifth aortic arch. METHODS: Four patients with persistent fifth aortic arch treated between July 1997 and June 2004 were included in this retrospective study. There were two girls and two boys, aged 2 days to 11.3 years, with persistent fifth aortic arch and cardiac conotruncal malformations. Chart recordings, plain chest films, two-dimensional and Doppler echocardiograms, cardiac catheterization with angiograms, surgical findings, and cytogenetic study were analyzed. RESULTS: Clinically, all four patients had the cardinal phenotypic features of 22q11.2 deletion syndrome, including cardiovascular malformations (conotruncal malformations and aortic arch anomalies), abnormal facies, thymic hypoplasia, canopy anomaly of the palate (high-arched palate, rather than cleft palate), and hypocalcemia (or hypoparathyroidism). All four patients were confirmed to have chromosome 22q11.2 deletion. CONCLUSION: Congenital conotruncal malformations, including tetralogy of Fallot with pulmonary atresia or stenosis, and aortic arch anomalies including a persistent fifth aortic arch or a right aortic arch, should lead to suspicion of chromosome 22q11.2 deletion when manifested together with any one of the other four cardinal phenotypic features.

A novel heterozygous missense mutation 377T > C (V126A) of TGIF gene in a family segregated with holoprosencephaly and moyamoya disease.

OBJECTIVES: To identify whether any mutations of candidate genes including SHH, ZIC2, SIX3, and TGIF exist in a Taiwanese family segregated with holoprosencephaly (HPE) and moyamoya disease. METHODS: Genotypes of the candidate genes SHH, ZIC2, SIX3, and TGIF were determined in the family members who were available for analysis by sequencing. In addition, genomic regions of another 50 unrelated Taiwanese (100 chromosomes) were studied to verify whether the nucleotide changes we found were mutations or polymorphisms. RESULTS: A novel missense mutation 377T > C and two polymorphisms (420A > G and 487C > T) in the TGIF gene were identified. No mutations in SHH, ZIC2 and SIX3 were found. The mother of the three HPE fetuses was found to be afflicted with moyamoya disease. A brief review of the mutations as well as polymorphisms reported in the TGIF gene up to 2005 is given. CONCLUSION: Molecular diagnosis can help genetic counseling in HPE, which is a heterogeneous disorder with its phenotypic and genotypic spectrum highly widened and variable. The possible association between TGIF mutation and moyamoya disease noted in our study also appeared to be novel.

Microchip, reverse transcription-polymerase chain reaction and culture methods to detect enterovirus infection in pediatric patients.

BACKGROUND: Enterovirus infection usually presents with mild and self-limited illness in children. However, Enterovirus type 71 can be characterized by neurotropism and may cause severe illness or even sudden death. Early detection of the virus will allow a physician to provide intensive or aggressive intervention. The purpose of the present study was to compare sensitivity of two innovative laboratory methods, that is, the DR.EV microchip method (DR. Chip Biotechnology, Shin-Tsu, Taiwan) and the reverse transcription-polymerase chain reaction (RT-PCR) method following conventional virus culture in detecting enterovirus infection in pediatric patients with herpangina or hand-foot-mouth disease. METHODS: A total of 87 children (age range: 1-8 years) were enrolled because of typical clinical findings of herpangina and hand-foot-mouth disease. Two hundred children selected after a careful clinical history review and physical examinations, were included as controls. All of these children had at least throat swab and rectal swab specimens taken and tested for evidence of enterovirus infection by microchip, RT-PCR and virus culture methods. In addition, 21 patients also had cerebrospinal fluid (CSF) specimens taken to test for possible central nervous system involvement. RESULT: The test results obtained from the 200 healthy kindergarten children were all negative for enteroviral infection by these three methods. Among the 87 test patients, the positive rates for throat swab, rectal swab and CSF by DR.EV chip, RT-PCR and virus culture were 71%, 68%, and 45% (throat swab); 66%, 61%, and 33% (rectal swab); and 52%, 29%, and 5% (CSF), respectively. There was no significant difference in the positive rates between the DR.EV chip and the RT-PCR methods (P > 0.1) on all types of specimens. However, statistically significant differences in positive rates were noted between the DR.EV chip and the conventional virus culture methods on all types of specimens (P < 0.001). Sensitivity of the microchip, RT-PCR and virus culture methods, was 82%, 72%, and 53%, respectively. CONCLUSION: The DR.EV chip method yielded a statistically higher positive rate and faster test results than the conventional viral culture method.

Antenatal treatment of chylothorax and cystic hygroma with OK-432 in nonimmune hydrops fetalis.

OBJECTIVES: To present our experience of using OK-432 in treating fetal cystic hygroma and chylothorax complicated with nonimmune hydrops fetalis. METHODS: OK-432 (Picibanil) was injected into the fetal pleural cavity or fetal cystic hygroma. RESULTS: Patient 1: A 23-year-old, gravida 2, para 1, was found to have a recurrent fetal chylothorax at GA 29 weeks. Serial amnioreduction and thoracocentesis was performed at GA 31, 32, 33, and 34 weeks. Intrapleural OK-432 injection was performed twice at GA 33 and 34 weeks. Cyanosis and respiratory distress were noted immediately after birth (GA 34 weeks). The baby expired despite of aggressive neonatal resuscitation. Patient 2: A 26-year-old, gravida 2, para 1, was found to have a cystic hygroma of her fetus at GA 17 weeks. Karyotype of the cystic fluid and the amniocytes were 46, XY. Fetal ascites developed at GA 22 weeks. OK-432 injection into the tumour was performed at GA 23 weeks. Stabilization of the cystic hygroma was noted throughout the pregnancy (about 3.5 cm in diameter). Serial fetal paracentesis and/or amnioreduction were performed. Karyotype of the ascites was again 46, XY. Maternal dietary modification with medium chain triglyceride was also prescribed. Chylothorax developed and the baby was born by cesareans at GA 32 weeks. Resolution of pleural effusion, ascites, and regression of cystic hygroma were noted since the 2nd day after birth. The baby had survived beyond 4 months of age at submission. CONCLUSION: Combination of antenatal OK-432 injection, maternal dietary modification, serial thoracocentesis plus paracentesis, together with amnioreduction and tocolysis, appeared to contribute to the success of antenatal treatment. Fetal pulmonary expansion may determine the immediate neonatal survival.

Maternal uniparental disomy in a patient with Prader-Willi syndrome with an additional small inv dup(15) chromosome.

Prader-Willi syndrome (PWS) is a complex genetic disorder. About 70% of cases have a paternal deletion at 15q11-q13, and most of the remaining cases are caused by maternal uniparental disomy (UPD). In rare cases of PWS with maternal UPD, small marker chromosomes are identified. Patients with inv dup(15) are at an increased risk of developing PWS or Angelman syndrome (AS) due to UPD. They may be also at increased risk for developmental delay due to additional copies of genes located within the PWS/AS critical region. Therefore, molecular investigations in patients with a supernumerary marker chromosome (SMC) are necessary to provide proper genetic counseling. We report a female infant with central hypotonia, weak crying, feeding problems, failure to thrive, and developmental delay after birth. Chromosome analysis revealed an SMC in 55% of metaphase cells. Fluorescence in situ hybridization showed that this marker chromosome was constituted by a small isodicentric inverted duplication of chromosome 15 [inv dup(15)]. Microsatellite analysis showed uniparental isodisomy of maternal chromosome 15 in the proband. Diagnosis of PWS was further confirmed by methylation-specific polymerase chain reaction. The inv dup(15) marker chromosome was also of maternal origin. Follow-up at the age of 18 months revealed a height in the 10th percentile and weight in the 50th percentile. She had poor activity and muscle tone, and was unable to walk independently. There was no psychomotor retardation, behavior disturbance or seizure.

Rapid detection of human HLA transgenes in pigs by fluorescence in situ hybridization (FISH) for adjuvant study of human xenotransplantation.

OBJECTIVES: We have recently generated several lines of transgenic pigs for HLA-DP and -DQ to elucidate the role of HLA-II antigens in the modulation of cell-mediated rejection of xenotransplantation. Using fluorescence in situ hybridization (FISH) analysis, the aim of this study was to determine integration sites and to test zygosity of these transgenes in the piglets after cross mating. METHODS: Blood lymphocytes of transgenic pigs for HLA-DP and -DQ were collected and cultured. Chromosome spreads were prepared by standard methodology. Gene constructs of HLA-DP A1+B1, -DQ A1 & B1 were labeled with fluorescein isothiocyanate or Texas Red by nick-translation. Hybridization was based on a standard FISH protocol. RESULTS: FISH analysis revealed that the HLA-DP probe hybridized to porcine chromosome 6, while both HLA-DQ A1 and B1 probes hybridized to porcine chromosome 11 at the same site. There was no cross-hybridization of HLA transgenes to the swine leukocyte antigen complex. Mosaic integration of HLA-DQ transgenes in the genome of F0, but full penetrance in F1 after selective breeding was observed. Both HLA-DP and HLA-DQ lines were determined to be heterozygous at the integration site. CONCLUSION: By FISH, we have detected specific integration sites of the HLA-DP and -DQ transgenes in pig genome and determined mosaic levels and zygosity types of these transgenes. We conclude that FISH is both sensitive and labor-efficient in confirming and differentiating transgenic pigs for multiple rejection-regulatory genes by visualizing individual integration sites in chromosomes or interphase nuclei.

Trisomy 13 mosaicism: study of serial cytogenetic changes in a case from early pregnancy to infancy.

We report a patient whose chorionic villus sampling showed a nonmosaic trisomy 13 [46,XX,der(13;13)(q10;q10)]. Subsequent amniocentesis and cordocentesis showed varying percentages of abnormal cells (77 and 78% in two amniocentesis; 14% in cordocentesis) and mosaic trisomy 13 was impressed. Prenatal fetal ultrasound scanning revealed only mild structural abnormalities (echogenic cardiac foci, transient lemon head, transient skin oedema). The mother chose to continue the pregnancy. Karyotyping of the cord blood, peripheral blood, umbilical cord, urine, and chorion were performed postpartum. The process of correction appeared to exist in the placenta (indirect evidence from coexistence of trisomy 13 [46,XX,der(13;13)(q10,q10)], euploidy [46,XX], aneuploidy [46,XX,-13, +mar], and monosomy 13 [45,XX,-13] in the chorion at birth). The baby had survived beyond eight months of age at the time of submission. Few structural abnormalities except low-set ears, absence of the 12th rib, and cardiomegaly with ventricular septal defect, were noted postnatally. The growth reached 95th percentile at the age of one month. Development milestones were not delayed at serial evaluations. Her ventricular septal defect was corrected surgically at the age of six months. Karyotypes of her skin fibroblasts, blood lymphocytes, and cardiac tissue were all normal [46,XX] at the time of surgery. Difficulties of the genetic counseling are also discussed.

Prenatal diagnosis of Machado-Joseph disease/Spinocerebellar Ataxia Type 3 in Taiwan: early detection of expanded ataxin-3.

Machado-Joseph disease (MJD)/Spinocerebellar Ataxia Type 3 (SCA3) is a rare autosomal dominative disorder in which one of the neurodegenerative disorders is caused by a translated CAG repeat expansion. Here, we present the first prenatal diagnosis of MJD in Taiwan in a woman whose husband was known to carry an unstable CAG repeat expansion in the MJD gene. After evaluating the couples' motivation and psychological tolerance, amniocentesis was performed at gestation of 13 weeks. The diagnosis was made using a simple nonradioactive polymerase chain reaction (PCR) for rapid detection of the presence of an expanded MJD allele. Meanwhile, using radioactive PCR, we identified the presence of an unusual shortness of CAG expansion in the MJD gene with 74 repeats in the fetus compared with 78 repeats in the father. After termination of the pregnancy, Western blot analysis further confirmed the presence of normal and mutant ataxin-3 in the fetal tissue. In summary, we have performed the first prenatal diagnosis of MJD in Taiwan, and described our experience with an at-risk male requesting counseling, carrier testing, and prenatal diagnosis for Machado-Joseph disease. Early detection of both normal and expanded ataxin-3 in fetal tissues was first demonstrated in the present study.

Detection of novel CFTR mutations in Taiwanese cystic fibrosis patients.

BACKGROUND AND PURPOSE: Cystic fibrosis (CF) in Asian populations is very rare. We performed molecular genetic analysis in 2 Taiwanese CF patients for detection of cystic fibrosis transmembrane conductance regulator (CFTR) mutations. METHODS: Temporal temperature gradient gel electrophoresis (TTGE) was used for mutation detection, and direct sequencing was used for identification of mutations. RESULTS: In one patient, 2 novel mutations, E7X and 989-992insA, were identified and the carrier status of his parents was confirmed. In the other patient, 3 mutations, S895N, 2215insG, and 1898+5G>T, were found. The 2215insG and S895N were found cis in the same chromosome. These splice site, frameshift, and nonsense mutations produce severely truncated CFTR polypeptides which lack a transmembrane domain, nucleotide binding folds, and the regulatory region, and are predicted to be null in CFTR function. CONCLUSIONS: These cases underscore the importance of comprehensive mutation analysis of Taiwanese CF patients. Definitive molecular findings can confirm the clinical diagnosis and facilitate patient management, carrier testing, and genetic counseling. Furthermore, there is an urgent need to understand the mutation spectrum and the clinical features of the CFTR gene in Asian patients in order that a mutation panel can be established for effective screening of CF chromosomes.