[Analysis of pathogenicity and genotype-phenotype correlation of the c.158G>A variant of phenylalanine hydroxylase gene].

OBJECTIVE: To explore the pathogenicity and genotype-phenotype correlation of the c.158G>A variant of phenylalanine hydroxylase (PAH) gene among patients with PAH deficiency. METHODS: Thirty seven children diagnosed with PAH deficiency at the Obstetrics and Gynecology Hospital Affiliated to Nanjing Medical University between July 2016 and June 2021 were selected as the study subjects. Clinical data and results of genetic testing were retrospectively analyzed. RESULTS: Among the 37 patients, mild hyperphenylalaninemia (HPA) was observed in 34 cases, two PAH variants (including c.158G>A), which formed a compound heterozygous mutation genotype, were detected in 33 patients, and the remainder one was found to harbor three PAH variants, including homozygous c.158G>A variants and a heterozygous c.842+2T>A variant. Classical phenylketonuria (PKU) was observed in 3 patients, and three PAH variants were detected in each of them, including two with c.[158G>A,842+2T>A]/c.728G>A and c.[158G>A,842+2T>A]/c.611A>G, respectively, and one with c.[158G>A, c.722G>A]/c.728G>A. The c.158G>A variant has a minimal influence on the PAH activity and is associated with a mild HPA phenotype. The variant should thereby be classified as likely benign. CONCLUSION: When the c.158G>A variant and other pathogenic variants are arranged in cis position, the ultimate phenotype will be determined by the pathogenicity of other variants.

Genetic analysis of a novel SUMF1 variation associated with a late infantile form of multiple sulfatase deficiency.

BACKGROUND: Multiple sulfatase deficiency (MSD) (MIM#272200) is an ultra-rare autosomal recessive lysosomal storage disorder caused by mutation of the Sulfatase Modifying Factor 1 (SUMF1) gene. METHODS: Herein, we report an eight-year-old boy with a late infantile form of multiple sulfatase deficiency. A combination of copy-number variation sequencing (CNV-seq) and whole-exome sequencing (WES) were used to analyze the genetic cause for the MSD patient. RESULTS: Our results, previously not seen in China, show a novel compound heterozygous mutation with one allele containing a 240.55 kb microdeletion on 3p26.1 encompassing the SETMAR gene and exons 4-9 of the SUMF1 gene, and the other allele containing a novel missense mutation of c.671G>A (p.Arg224Gln) in the SUMF1 gene. Both were inherited from the proband's unaffected parents, one from each. Bioinformatics analyses show the novel variation to be "likely pathogenic." SWISS-MODEL analysis shows that the missense mutation may alter the three-dimensional (3D) structure. CONCLUSIONS: In summary, this study reported a novel compound heterozygous with microdeletion in SUMF1 gene, which has not been reported in China. The complex clinical manifestations of MSD may delay diagnosis; however, molecular genetic analysis of the SUMF1 gene can be performed to help obtain an early diagnosis.

[Clinical features and genetic variants of a case with carnitine palmitoyltransferase 1A deficiency].

OBJECTIVE: To identify the possible pathogenesis of a neonate with carnitine palmitoyltransferase 1A (CPT1A) deficiency by analyzing gene variants. METHODS: Potential variants were detected with an Ion Torrent semiconductor sequencer using a gene panel for inherited diseases, and gene variants were verified by Sanger sequencing. RESULTS: Genetic testing indicated that the neonate has carried c.1895T>A(p.Leu632X) and c.1153G>A (p.Ala385Thr) compound heterozygous variants of the CPT1A gene, which were inherited from his father and mother, respectively. Both variants were verified as novel through the retrieval of HGMD database, ClinVar database and literature. According to the standards and guidelines of the American College of Medical Genetics and Genomics, the c.1895T>A variant was predicted to be pathogenic (PVS1+PM2+PP4) and c.1153G>A as likely pathogenic (PM1+PM2+PM3+PP3). CONCLUSION: The c.1895T>A and c.1153G>A compound heterozygous variants of the CPT1A gene might underlie the pathogenesis of this child. Above results have provided a basis for clinical diagnosis and genetic counseling, and enriched the variant spectrum of the CPT1 deficiency.

[Tandem mass spectrometry and genetic variant analysis of four neonates with very long chain acyl-coenzyme A dehydrogenase deficiency].

OBJECTIVE: To analyze the clinical features and genetic variants in four neonates with very long chain acyl-coenzyme A dehydrogenase (VLCAD) deficiency. METHODS: Neonates with a tetradecenoylcarnitine (C14:1) concentration at above 0.4 µmol/L in newborn screening were recalled for re-testing. Four neonates were diagnosed with VLCAD deficiency by MS-MS and genetic testing, and their clinical features and genotypes were analyzed. RESULTS: All cases had elevated blood C14:1, and the values of first recalls were all lower than the initial test. In 2 cases, the C14:1 had dropped to the normal range. 1 case has remained at above 1 µmol/L after the reduction, and the remainder one case was slightly decreased. In total eight variants of the ADACVL genes were detected among the four neonates, which included 5 missense variants and 3 novel variants (p.Met344Val, p.Ala416Val, c.1077+6T>A). No neonate showed salient clinical manifestations. CONCLUSION: Above findings have enriched the spectrum of ADACVL gene mutations and provided a valuable reference for the screening and diagnosis of VLCAD deficiency.

A joint method for the screening of pharmacological chaperones for phenylalanine hydroxylase.

Phenylalanine hydroxylase (PAH) deficiency (PAHD) is an autosomal recessive disorder that causes severe injury to the nervous system, the treatment of which mainly depends on dietary therapy. The limited treatment options for PAHD are an incentive to develop new methods to identify more efficient therapeutic drugs, such as agonists which could improve PAH activity. In this study, we aimed to establish a rapid and convenient method for the screening and verification of PAH agonists. We compared fluorospectrophotometry and tandem mass spectrometry for detection of enzymatic formation of tyrosine, finding that the latter was a more sensitive method. We optimized immunoprecipitation purification conditions and measurement conditions of PAH activity. The optimal ratio between PAH protein and magnetic beads was 500 µg protein per 20 µL beads, and the optimized conditions for the detection of PAH enzymatic activity included the presence of 75 µM coenzyme ((6R)-l-erythro-5,6,7,8-tetrahydrobiopterin) and 30 min reaction time. Based on virtual screening, we screened ten candidate agonists from the FDA drug library. Three of these (nefopam, fluocinonide, and risperidone) were found to activate the enzyme in a dose-dependent manner (0.1-10 µM) by the joint method. We tested the efficacy of the three agonists on three PAH mutations (p.I65T, p.H107R, and p.D101N) that influence enzyme activity, and found that risperidone could specifically activate D101N-mutated enzyme. In conclusion, we established a joint method that is highly reliable, cost-effective, labor-saving, and time-saving. And we also found a specific agonist for D101N-mutated PAH by this joint method which may assist the development of clinical treatment for PAHD patients with different enzyme deficiencies.

Genetic analysis of 62 Chinese families with Duchenne muscular dystrophy and strategies of prenatal diagnosis in a single center.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a severe X-linked recessive neuromuscular disorder. Patients with DMD usually have severe and fatal symptoms, including progressive irreversible muscle weakness and atrophy complicated with gastrocnemius muscle pseudohypertrophy. DMD is caused by mutations in the dystrophin-encoding DMD gene, including large rearrangements and point mutations. This retrospective study was aimed at supplying information on our 4-year clinical experience of DMD genetic and prenatal diagnosis at the Department of Prenatal Diagnosis in Women's Hospital of Nanjing Medical University. METHODS: Multiplex ligation-dependent probe amplification (MLPA) was used to detect the exon deletions or duplications. And Ion AmpliSeq™ panel for inherited disease was used as the next-generation sequencing (NGS) method to identify the point mutations in exons of DMD gene, but the introns were not sequenced. RESULTS: In this study, the large deletions and duplications of DMD gene were detected in 32 (51.6%) of the 62 families, while point mutations were detected in 20 families (32.3%). The remaining 10 families with a negative genetic diagnosis need to be reevaluated for clinical symptoms or be detected by other molecular methods. Notably, six novel mutations were identified, including c.412A > T(p.Lys138*), c.2962delT(p.Ser988Leufs*16), c.6850dupA (p.Ser2284Lysfs*7), c.5139dupA (p.Glu 1714Argfs*5), c.6201_6203delGCCins CCCA(p.Val2069Cysfs*14) and c.10705A > T (p.Lys3569*). In 52 families with positive results, 45 mothers (86.5%) showed positive results during carrier testing and de novo mutations arose in 7 probands. The prenatal diagnosis was offered to 34 fetuses whether the pregnant mother was a carrier or not. As a result, eight male fetuses were affected, three female fetuses were carriers, and the remaining fetuses had no pathogenic mutation. CONCLUSIONS: This study reported that MLPA and NGS could be used for screening the DMD gene mutations. Furthermore, the stepwise procedure of prenatal diagnosis of DMD gene was shown in our study, which is important for assessing the mutation type of fetuses and providing perinatal care in DMD high-risk families.

[Clinical and genetic analysis of two children suspected for argininosuccinic aciduria].

OBJECTIVE: To analyze the clinical and genetic features of two children suspected for arginylsuccinuria aciduria. METHODS: The patients were subjected to high-throughput sequencing using a gene panel. RESULTS: Both patients had high citrulline (87.37-156.10 µmol/L) measured by mass spectrometry/mass spectrometry (MS/MS) upon neonatal screening but had no symptoms. Two compound heterozygous variants of the ASL gene were detected in patient 1 (exon 6: c.467C>T inherited from her father and exon 7: c.556C>T inherited from her mother), among which c.556C>T is novel. Patient 2 had mental retardation and two full siblings who had died of hyperammonemia. Two compound heterozygosity variants of the ASL gene were detected (exon 3: c.281G>T inherited from his father and intron: c.208-15T>A inherited from his mother). Both were novel mutations. CONCLUSION: Variants of the ASL gene probably underlie the argininosuccinic aciduria in the two patients. Above findings have enriched the spectrum of ASL mutations.

[Analysis of AGR1 gene variants in an infant with early-onset argininemia].

OBJECTIVE: To explore the genetic basis for an infant with early-onset argininemia. METHODS: Potential variant was detected with an Ion Torrent semiconductor sequencer using a gene panel for inherited diseases. Suspected variants were verified by Sanger sequencing. RESULTS: Genetic testing indicated that he has carried c.560+2T>C and c.811T>C compound heterozygous variant of the AGR1 gene, which were inherited from his father and mother, respectively. Among these, c.560+2T>C was suspected to be pathogenic, while c.811T>C was of unknown clinical significance, and both were not reported previously. CONCLUSION: The c.560+2T>C and c.811T>C compound heterozygous variants of the AGR1 gene probably underlies the argininemia in this child. Above finding has enriched the variant spectrum of the AGR1 gene.

Identification and characterization of a novel 43-bp deletion mutation of the ATP7B gene in a Chinese patient with Wilson's disease: a case report.

BACKGROUND: Wilson's disease (WD) is an autosomal recessive disorder characterized by copper accumulation. ATP7B gene mutations lead to ATP7B protein dysfunction, which in turn causes Wilson's disease. CASE PRESENTATION: We describe a male case of Wilson's disease diagnosed at 10 years after routine biochemical test that showed low serum ceruloplasmin levels and Kayser-Fleischer rings in both corneas. Analysis of the ATP7B gene revealed compound heterozygous mutations in the proband, including the reported c.3517G > A mutation and a novel c.532_574del mutation. The c.532_574del mutation covered a 43-bp region in exon 2, and resulted in a frameshift mutation (p.Leu178PhefsX10). By base sequence analysis, two microhomologies (TCTCA) were observed on both deletion breakpoints in the ATP7B gene. Meanwhile, the presence of some sequence motifs associated with DNA breakage near the deletion region promoted DNA strand break. CONCLUSIONS: By comparison, a replication-based mechanism named fork stalling and template switching/ microhomology-mediated break-induced replication (FoSTeS/MMBIR) was used to explain the formation of this novel deletion mutation.

Prenatal chromosomal microarray analysis in fetuses with congenital heart disease: a prospective cohort study.

BACKGROUND: Currently, chromosomal microarray analysis is considered the first-tier test in pediatric care and prenatal diagnosis. However, the diagnostic yield of chromosomal microarray analysis for prenatal diagnosis of congenital heart disease has not been evaluated based on a large cohort. OBJECTIVE: Our aim was to evaluate the clinical utility of chromosomal microarray as the first-tier test for chromosomal abnormalities in fetuses with congenital heart disease. STUDY DESIGN: In this prospective study, 602 prenatal cases of congenital heart disease were investigated using single nucleotide polymorphism array over a 5-year period. RESULTS: Overall, pathogenic chromosomal abnormalities were identified in 125 (20.8%) of 602 prenatal cases of congenital heart disease, with 52.0% of them being numerical chromosomal abnormalities. The detection rates of likely pathogenic copy number variations and variants of uncertain significance were 1.3% and 6.0%, respectively. The detection rate of pathogenic chromosomal abnormalities in congenital heart disease plus additional structural anomalies (48.9% vs 14.3%, P < .0001) or intrauterine growth retardation group (50.0% vs 14.3%, P = .044) was significantly higher than that in isolated congenital heart disease group. Additionally, the detection rate in congenital heart disease with additional structural anomalies group was significantly higher than that in congenital heart disease with soft markers group (48.9% vs 19.8%, P < .0001). No significant difference was observed in the detection rates between congenital heart disease with additional structural anomalies and congenital heart disease with intrauterine growth retardation groups (48.9% vs 50.0%), congenital heart disease with soft markers and congenital heart disease with intrauterine growth retardation groups (19.8% vs 50.0%), or congenital heart disease with soft markers and isolated congenital heart disease groups (19.8% vs 14.3%). The detection rate in fetuses with congenital heart disease plus mild ventriculomegaly was significantly higher than in those with other types of soft markers (50.0% vs 15.6%, P < .05). CONCLUSION: Our study suggests chromosomal microarray analysis is a reliable and high-resolution technology and should be used as the first-tier test for prenatal diagnosis of congenital heart disease in clinical practice.

Perinatal outcomes following cell-free DNA screening in >32 000 women: Clinical follow-up data from a single tertiary center.

OBJECTIVE: Cell-free DNA (cfDNA) screening for aneuploidy was clinically introduced in 2011. We aim to focus on the follow-up information from a single tertiary center undergoing genome-wide cfDNA screening to evaluate this technology. METHOD: A total of 32 431 cases were retrospectively reviewed. The screening was performed using a BGI protocol, and the cfDNA results were analyzed together with the pregnancy outcomes, confirmatory testing results, and ultrasound findings. RESULTS: Of the 32 431 cfDNA screening cases, successful follow-up was conducted in 287 (82.2%) cases with high-risk cfDNA results, 85 (94.4%) cases with copy number variation (CNV) and rare autosomal trisomy (RAT) results, and 26 060 (81.5%) cases with low-risk cfDNA results. Among them, 234 with high-risk cfDNA results chose invasive testing, revealing 169 true positive cases. In cases with CNV and RAT results, 45 cases underwent invasive diagnosis, revealing six pathogenic CNVs and three uniparental disomies. In cases with low-risk cfDNA results, three false negative cases were confirmed. CONCLUSION: Cell-free DNA screening appears to be effective in detecting the common autosomal aneuploidies, but one-third of our cohort with high-risk results rejected confirmatory testing. Our data provide information on the clinical experience of large-scale whole-genome cfDNA screening that has global relevance for the implementation of this technology.

[Clinical report of testicular hypoplasia combined with 21-hydroxylase deficiency].

OBJECTIVE To investigate the correlation of 21-hydroxylase deficiency (21-OHD) with male testicular dysplasia. METHODS Clinical data of 8 infertile males with congenital adrenal hyperplasia due to 21-OHD was retrospectively analyzed. In addition, potential mutations of the CYP21A2 gene was detected. RESULTS All patients were referred because of azoospermia or severe oligospermia and had small testis with averaged testicular volume of 6.1 mL. Three patients had testicular adrenal rest tumors. Endocrinologic examinations revealed low levels of leutinizing hormone and follicular stimulating hormone, normal or elevated testosterone, elevated progesterone, elevated or normal adrenocoticotropic hormone, and low or normal cortisol. All patients had adrenal cortical hyperplasia, 5 with adrenal adenoma, 1 case associated with bilateral adrenal myelolipoma. All patients were given glucocorticoid replacement therapy for 3 to 6 months, which successfully improved the seminal status of 6 patient and resulted pregnancies in 5 couples. Seven pathogenic mutations of the CYP21A2 gene among the 8 patients. CONCLUSION 21-OHD can cause testicular hypoplasia and spermatogenic failure. Glucocorticoids and operations can obtain good result and improve spermatogenesis. Our results have shown a good genotype/phenotype correlation in these cases. All patients have carried the p.Ile172Asn mutation, which is associated with simple virilizing form.

[Genetic analysis and prenatal diagnosis for 25 Chinese pedigrees affected with congenital adrenal hyperplasia due to 21-hydroxylase deficiency].

OBJECTIVE: To identify pathogenic mutations in 25 Chinese pedigrees affected with congenital adrenal hyperplasia (CAH). METHODS: Mutations of the CYP21A2 gene were detected with locus-specific PCR/restriction endonuclease analysis, multiplex ligation-dependent probe amplification assay, and direct sequencing of the entire CYP21A2 gene. Prenatal diagnosis was offered to fetuses at risk for CAH. RESULTS: All 50 alleles of the CYP21A2 gene carried by the 25 pedigrees were successfully delineated. Large deletions and conversions have accounted for 16 (32%) of the alleles, which included 9 entire CYP21A2 gene deletions, 6 chimeric CYP21A1P/CYP21A2 genes, and 1 partial conversion of the CYP21A2 gene. For the remaining 34 alleles, there were 9 micro-conversions and 4 de novo mutations [including a previously unreported c.62G>A (p.Trp21X) mutation]. Prenatal diagnosis was provided for 28 fetuses with a high risk for CAH, among whom 8 were found to be affected. CONCLUSION: The detection of CYP21A2 gene mutations can facilitate appropriate genetic counseling and prenatal diagnosis for the affected pedigrees.

[Analysis of MAT1A gene mutations in a child affected with simple hypermethioninemia].

OBJECTIVE: To detect potential mutations of MAT1A gene in a child suspected with simple hypermethioninemia by MS/MS neonatal screening. METHODS: Clinical data of the child was collected. Genomic DNA was extracted by a standard method and subjected to targeted sequencing using an Ion AmpliseqTM Inherited Disease Panel. Detected mutations were verified by Sanger sequencing. RESULTS: The child showed no clinical features except evaluated methionine. A novel compound mutation of the MAT1A gene, i.e., c.345delA and c.529C>T, was identified in the child. His father and mother were found to be heterozygous for the c.345delA mutation and c.529C>T mutation, respectively. CONCLUSION: The compound mutation c.345delA and c.529C>T of the MAT1A gene probably underlie the disease in the child. The semi-conductor sequencing has provided an important means for the diagnosis of hereditary diseases.

[Prenatal diagnosis of two fetuses with chromosome 1p36 deletion syndrome].

OBJECTIVE: To analyze two fetuses with multiple malformations revealed by ultrasonography using single nucleotide polymorphism array (SNP array), and to explore the strategy for the prenatal diagnosis of 1p36 deletion syndrome. METHODS: Amniocentesis was performed on the two pregnant women. Amnion fluid cells were cultured, and karyotypes of the fetuses were determined through G-banding analysis. Whole genome SNP array was used to detect genomic anomalies of the two fetuses. The karyotypes of their parents were determined through G-banding analysis of peripheral venous blood samples. RESULTS: G-banding analysis showed a 46,XY,add(1p36)? and a 46,XX,add(1p36)? karyotype for fetuses 1 and 2, respectively. SNP array analysis showed that the fetus 1 had arr[19]1p36.33p36.32 (752 566 - 3 393 462)×1 and 7q35q36.3 (144 480 549 - 159 119 486)×3, and fetus 2 had arr[19]1p36.33p36.23 (752 566 - 8 362 754)×1, 6p25.3p22.3 (204 909 - 20 182 185)×3. The mother of fetus 1 had a 46,XX,t(1;7)(p36;q35) karyotype, and the mother of fetus 2 had a 46,XX,t(1;6)(p36;p22) karyotype. The karyotypes of both fathers appeared to be normal. CONCLUSION: SNP array has the advantages such as high sensitivity and high accuracy for prenatal diagnosis, and can provide more detailed information for genetic counseling of 1p36 deletion syndrome.

[Detection of TSC1/TSC2 gene mutations among patients with tuberous sclerosis complex by Ion Torrent semiconductor sequencing].

OBJECTIVE: To develop and validate a method for mutation screening and prenatal diagnosis of TSC1/TSC2 mutations among patients with tuberous sclerosis complex (TSC) by Ion Torrent semiconductor sequencing. METHODS: Potential mutations of SC1/TSC2 gene was detected in 2 TSC families and 1 sporadic TSC patient using an Ion Torrent PGM sequencer. Candidate variants were validated by Sanger sequencing. The corresponding site of TSC2 in the fetus of family 2 was also detected with Sanger sequencing. RESULTS: Ion Torrent semiconductor sequencing has identified a probably pathogenic TSC2 mutation (c.311-312insGCTG) in the patient from family 1, and a probably pathogenic TSC2 mutation (c.1790A>G) in the patient of family 2. CONCLUSION: Targeted Ion Torrent PGM sequencing is an accurate and efficient method to detect TSC1/TSC2 mutations in TSC.

[Mutation screening and prenatal diagnosis of methylmalonic academia in a Chinese pedigree by Ion Torrent semiconductor sequencing].

OBJECTIVE: To identify pathogenic mutations in a Chinese pedigree affected with methylmalonic academia for genetic counseling and prenatal diagnosis. METHODS: Molecular analysis of the MUT, MMACHC, MMAA and MMAB genes was performed for the proband with methylmalonic academia by Ion Torrent semiconductor sequencing. Candidate mutations were validated by Sanger sequencing. The couple was offered prenatal diagnosis via analyzing of the fetal DNA through amniocentesis. RESULTS: The proband was found to be compound heterozygous for c.609G>A (p.Trp203X) and c.658-660del AAG (p.Lys220del) mutations, which were inherited respectively from each of his parents. Prenatal diagnosis showed that the fetus has inherited two wild-type parental alleles. CONCLUSION: The targeted Ion Torrent PGM sequencing has detected pathogenic mutations in the Chinese pedigree affected with methylmalonic academia, which has provided molecular evidence for clinical diagnosis, genetic counseling and prenatal diagnosis for the family.

Molecular characterization of ring chromosome 18 by low-coverage next generation sequencing.

BACKGROUND: Ring chromosomes are one category of structurally abnormal chromosomes that can lead to severe growth retardation and other clinical defects. Traditionally, their diagnosis and characterization has largely relied on conventional cytogenetics and fluorescence in situ hybridization, array-based comparative genomic hybridization and single nucleotide polymorphism array-based comparative genomic hybridization. However, these methods are ineffectively at characterizing the ring chromosome structure and only offer a low resolution mapping of breakpoints. Here, we applied whole-genome low-coverage paired-end next generation sequencing (NGS) to two suspected cases of ring chromosome 18 (r(18)) and characterized the ring structure including the chromosome dosage changes and the breakpoint junction. METHODS: The breakpoints and chromosome copy number variations (CNVs) of r(18) were characterized by whole-genome low-coverage paired-end NGS. We confirmed the dosage change by single nucleotide polymorphisms array, and validated the junction site regions using PCR followed by Sanger sequencing. RESULTS: We successfully and fully characterized the r(18) in two cases by NGS. We mapped the breakpoints with a high resolution and identified all CNVs in both cases. We analyzed the breakpoint regions and discovered two breakpoints located within repetitive sequence regions, and two near the repetitive sequence regions. One of the breakpoints in case 2 was located within the gene METTL4, while the other breakpoints were intergenic. CONCLUSIONS: We demonstrated that whole-genome low-coverage paired-end NGS can be used directly to map breakpoints with a high molecular resolution and detect all CNVs on r(18). This approach will provide new insights into the genotype-phenotype correlations on r(18) and the underlying mechanism of ring chromosomes formation. Our results also demonstrate that this can be a powerful approach for the diagnosis and characterization of ring chromosomes in the clinic.

[Detection of pathogenic mutations for methylmalonic acidemia using new-generation semiconductor targeted sequencing].

OBJECTIVE: To detect the pathogenic mutation in a patient with methylmalonic acidemia using IonTorrent Personal Genome Machine (PGM) and assess the feasibility of such technology for analyzing complex monogenic diseases. METHODS: Peripheral blood sample was collected from the patient. Genomic DNA was isolated using a standard method and subjected to targeted sequencing using an Ion Ampliseq Inherited Disease Panel. DNA fragment was ligated with a barcoded sequencing adaptor. Template preparation, emulsion PCR, and Ion Sphere Particles enrichment were carried out using the Ion One Touch system. Data from the PGM runs were processed using Ion Torrent Suite 3.2 software to generate sequence reads. All variants were filtered against dbSNPl37. DNA sequences were visualized with an Integrated Genomics Viewer. RESULTS: After data analysis and database filtering, a previously reported nonsense mutation, c.586C>T (p.R196X), and a novel mutation c.898C>T (p.R300X) were identified in the MMAA gene in this patient. Both mutations were verified by conventional Sanger sequencing. CONCLUSION: Pathogenic MMAA mutations have been identified in a patient with methylmalonic acidemia. This new-generation targeted sequencing on the PGM sequencers can be applied for genetic diagnosis of hereditary diseases.

[Application of multiplex ligation-dependent probe amplification for rapid detection of aneuploidies and structural chromosomal abnormalities in prenatal diagnosis].

OBJECTIVE: To explore the value of multiplex ligation-dependent probe amplification (MLPA) for rapid detection of aneuploidies and structural chromosomal abnormalities during prenatal diagnosis. METHODS: Two hundred and eight six amniotic fluid samples were analyzed with both MLPA and conventional karyotyping. Structural abnormalities were verified with array comparative genomic hybridization. RESULTS: Ten cases of trisomy 21, 2 cases of trisomy 18, 1 case of trisomy 13, 1 case of mosaic trisomy 21, 1 case of 45,X, 1 case of large deletion of Xp, 1 case of trisomy 18p and 1 case of large deletion of 18p and 18q were identified. The same results were derived by both MLPA and conventional karyotyping. Structural abnormalities were verified by array comparative genomic hybridization (aCGH) with 100% accuracy. CONCLUSION: In addition to aneuploidies, MLPA can rapidly identify large deletions and duplications of chromosomes 21, 18, 13, X and Y. MLPA is supplementary to conventional karyotyping for identification of such chromosomal abnormalities prenatal diagnosis.