Mei-Hui Lee

Two novel null mutations in a Taiwanese cystic fibrosis patient and a survey of East Asian CFTR mutations

Cystic fibrosis (CF; OMIM number 219700) is one of the most common and life-shortening autosomal recessive disorders in Caucasians, with an incidence of 1 in 2,500 newborns [Welsh et al., 2001]. Its incidence is significantly reduced in Asian populations (1 in 90,000) [Wright and Morton, 1968]. Little is known about the spectrum of CFTR mutations in Asians. Data from the small number of reports indicated that a potentially large pool of Asian CF mutations are novel. The commercially available mutation panels are not suitable for screening Asian CF mutations. A more comprehensive mutation detectionmethod is necessary to search for the unknown mutations. Using recently developed temporal temperature gradient gel electrophoresis (TTGE) method [Wong et al., 2001; Alper et al., in press (to be published elsewhere)], a CF patient from Taiwan was found to be compound heterozygote for E7X and 989992insA novel null mutations. The patient was a 11⁄2-year-old boy who was born at 41 weeks of gestation with normal weight and unremarkablemedical history until 7months of agewhen he was admitted to hospital in Taiwan with severe vomiting and abdominal distension. He was diagnosed with acute gastroenteritis. At 1 year of age, he had severe cough, poor appetite, and sputum colonization with Pseudomonas aeruginosa. The diagnosis of CF was made when the patient was 2 years old, based on the recurrent respiratory tract infection with pneumonia, sputum colonization with Pseudomonas and Staphylococcus epidermis, mucosal pluggings, bronchiectasis, echogenic bowl, developmental delay, steatorrhea, and elevated sweat chloride (89mmol/L).Molecular analysis by Genzyme Genetics pan-ethnic 87 mutation panel did not reveal anyCFTRmutations. Since autopsy revealed evidence of CF in his deceased older sister, a comprehensive mutational analysis of the CFTR gene was felt necessary. CFTR exons and their flanking intron regions were amplified using 27 primer pairs followed by TTGE analysis [Wong et al., 2001]. Exons 1 and 6b that revealed abnormal TTGE banding patterns were sequenced (data not shown). Twomutationswere identified;G151T in exon 1 changes a glutamate at amino acid residue 7 to a stop codon, and 989-992 insertion A causes frameshift and a truncated CFTR protein of 306 amino acids. The intron 8 polythymidine and GT tract variants were found to be homozygous 7T and heterozygous 11/12 GT, both were unremarkable. Although the parents were unavailable for carrier testing, the finding of two deleterious mutations and the positive family history substantiates the diagnosis of CF in this patient. Unfortunately, the patient died before the mutations were identified. He was pancreatic insufficient. Since CF cases were rarely seen in Taiwan, the patient was not properly treated with enzyme supplement. The two mutations found in our patient have never been reported. The patient’s father is from Taiwan and the mother from Vietnam. A literature search found more than 80 documented cases of CF patients in East Asia [Macek et al., 1992; Wang et al., 1993; Crawford et al., 1995; Zielenski et al., 1995a; Hojo et al., 1997; Macek et al., 1997; Suwanjutha et al., 1998; Seki et al., 1999; Wagner et al., 1999; Yoshimura et al., 1999; Morokawa et al., 2000; Wu et al., 2000]. Approximately 50 caseswere reported in Japan and about 10 cases have been reported in patients from Taiwan and Mainland China [Seki et al., 1999]. Most of the patients have not been analyzed at themolecular level. Only 15 (including this study) had identified CFTR mutations (Table I). It is remarkable that the East Asian CF patients do not share any mutations with patients of other ethnic backgrounds. Even within the East Asians, the CFTR mutation spectrum of Chinese patients is distinct from that of Japanese patients (Table I) [Hojo et al., 1997; Macek et al., 1997; Seki et al., 1999; Yoshimura et al., 1999; Morokawa et al., 2000]. Most of the mutations are frameshift, splice sitemutations, or nonsensemutations that predict a CFTR protein with null activity. All patients presented classical CFphenotype (Table I)with elevated sweat chloride and pancreatic insufficiency. *Correspondence to: Dr. Lee-Jun C. Wong, Ph.D., Associate Professor, Institute for Molecular and Human Genetics, P.O. Box 571477, Georgetown University Medical Center, 3800 Reservoir Rd, NW M4000, Washington, DC 20057. E-mail: wonglj@georgetown.edu

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.

A case of restrictive dermopathy with complete chorioamniotic membrane separation caused by a novel homozygous nonsense mutation in the ZMPSTE24 gene.

A Case of Restrictive Dermopathy With Complete Chorioamniotic Membrane Separation Caused by a Novel Homozygous Nonsense Mutation in the ZMPSTE24 Gene Ming Chen, Hsiang-Hsu Kuo, Yi-Chen Huang, Yu-Yuan Ke, Shun-Ping Chang, Chih-Ping Chen, Dong-Jay Lee, Meng-Luen Lee, Mei-Hui Lee, Tze-Ho Chen, Chia-Hsiang Chen, Hui-Mei Lin, Chin-San Liu, and Gwo-Chin Ma* Department of Genomic Medicine, Changhua Christian Hospital, Changhua, Taiwan Department of Medical Research, Changhua Christian Hospital, Changhua, Taiwan Department of Obstetrics and Gynecology, Changhua Christian Hospital, Changhua, Taiwan Department of Obstetrics and Gynecology, National Taiwan University Hospital, Taipei, Taiwan Department of Medical Genetics, National Taiwan University Hospital, Taipei, Taiwan Department of Obstetrics and Gynecology, Puli Christian Hospital, Nantou, Taiwan Department of Pediatrics, Puli Christian Hospital, Nantou, Taiwan Department of Pediatrics, Changhua Christian Hospital, Changhua, Taiwan Department of Obstetrics and Gynecology, Mackay Memorial Hospital, Taipei, Taiwan Department of Medical Research, Mackay Memorial Hospital, Taipei, Taiwan Department of Neurology, Vascular Biology and Genomics Center, Changhua Christian Hospital, Changhua, Taiwan Institute of Biochemistry and Biotechnology, Chung-Shan Medical University, Taichung, Taiwan

Preimplantation and prenatal genetic diagnosis of aromatic L-amino acid decarboxylase deficiency with an amplification refractory mutation system-quantitative polymerase chain reaction

OBJECTIVES To develop a diagnostic platform for preimplantation genetic diagnosis (PGD) and prenatal genetic diagnosis (PND) to prevent births of aromatic L-amino acid decarboxylase deficiency (AADC) patients. MATERIALS AND METHODS Five Taiwanese families carrying AADC were enrolled. A novel technique, amplification refractory mutation system-quantitative polymerase chain reaction (ARMS-qPCR), was developed for both of PGD and PND. For PGD, blastomere biopsies of day-3 cleavage-stage embryos were subjected to ARMS-qPCR. Villi, cultured amniocytes, or both were used to confirm the PGD result; this approach could also be used as the sole method for PND after in vivo conception). RESULTS Unaffected live births were achieved in four of the five families, except one with ongoing PGD. The ARMS-qPCR correctly classified blastomeres (from day-3 cleavage-stage embryos) as affected (homozygous mutant), carrier (heterozygous for mutant and wild-type alleles), or normal (homozygous wild-type) within 1 working day. CONCLUSIONS To our knowledge, this is the first report of successful PGD of AADC. The molecular technique we devised (ARMS-qPCR) was applicable for PGD as well as PND of AADC. Furthermore, it has great potential for similar applications in other monogenic disorders.

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.

The use of low molecular weight heparin reduced the fetal fraction and rendered the cell-free DNA testing for trisomy 21 false negative

Cell-free DNA (cfDNA) screening for fetal trisomy 21, 18, and 13 is now widely used in clinical practice. However, false-positives and false-negatives did occur1,2. Actually, the majority of fetal cfDNA in maternal blood is from placental trophoblasts.Cell-free DNA (cfDNA) screening for fetal trisomy 21, 18, and 13 is now widely used in clinical practice. However, false-positives and false-negatives did occur1,2. Actually, the majority of fetal cfDNA in maternal blood is from placental trophoblasts.

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.

Normal prenatal ultrasound findings reflect outcome in case of trisomy 14 confined placental mosaicism developing after preimplantation genetic diagnosis

Preimplantation genetic diagnosis (PGD) is a well-established technology that is widely applied in assisted reproductive medicine. However, misdiagnosis by PGD can still occur and thus invasive prenatal diagnosis may be necessary1. Until recently, non-invasive cell-free DNA (cfDNA) testing was advocated for confirmation of a PGD result. However, in certain situations, for example, when there is confined placental mosaicism (CPM), results of cfDNA testing might not correlate with the clinical phenotype2. We describe a case of a normal liveborn baby with a prenatal karyotype of mosaic trisomy 14, despite being conceived after PGD. Detailed prenatal genetic assessments, together with frequent ultrasound examinations, indicated a possible case of CPM, which was confirmed by postnatal diagnosis of the neonate and placenta. The timeline of our investigations is illustrated in Figure 1.

Normal prenatal ultrasound findings reflect outcome in case of trisomy 14 confined placental mosaicism developing after preimplantation genetic diagnosis: Mosaic trisomy 14 originated from mitotic error despite PGD

Preimplantation genetic diagnosis (PGD) is a well-established technology that is widely applied in assisted reproductive medicine. However, misdiagnosis by PGD can still occur and thus invasive prenatal diagnosis may be necessary1. Until recently, non-invasive cell-free DNA (cfDNA) testing was advocated for confirmation of a PGD result. However, in certain situations, for example, when there is confined placental mosaicism (CPM), results of cfDNA testing might not correlate with the clinical phenotype2. We describe a case of a normal liveborn baby with a prenatal karyotype of mosaic trisomy 14, despite being conceived after PGD. Detailed prenatal genetic assessments, together with frequent ultrasound examinations, indicated a possible case of CPM, which was confirmed by postnatal diagnosis of the neonate and placenta. The timeline of our investigations is illustrated in Figure 1.

Ultrasonography for prognosis in case of trisomy 14 confined placental mosaicism developing after preimplantation genetic screening

Preimplantation genetic diagnosis (PGD) is a well-established technology that is widely applied in assisted reproductive medicine. However, misdiagnosis by PGD can still occur and thus invasive prenatal diagnosis may be necessary1. Until recently, non-invasive cell-free DNA (cfDNA) testing was advocated for confirmation of a PGD result. However, in certain situations, for example, when there is confined placental mosaicism (CPM), results of cfDNA testing might not correlate with the clinical phenotype2. We describe a case of a normal liveborn baby with a prenatal karyotype of mosaic trisomy 14, despite being conceived after PGD. Detailed prenatal genetic assessments, together with frequent ultrasound examinations, indicated a possible case of CPM, which was confirmed by postnatal diagnosis of the neonate and placenta. The timeline of our investigations is illustrated in Figure 1.