Cell-free DNA test for pathogenic copy number variations: A retrospective study.

OBJECTIVE: To evaluate the detection rate (DR) by prenatal cell-free DNA test for pathogenic copy number variations (CNVs)＞2 Mb among pregnancies with fetal ultrasound abnormalities. MATERIALS AND METHODS: This was a retrospective study on 29 pregnant women with fetuses diagnosed as microdeletion/microduplication syndromes by prenatal chromosome microarray analysis (CMA). Cell-free DNA from the maternal plasma was sequenced on the NextSeq CN500 sequencer. The quality standard of unique map reads in a single sample was greater than 10 M and only gains and losses of more than 2 Mb were reported. RESULTS: A total of 24 CNVs were identified by cell-free DNA test among the 21 fetuses with pathogenic CNVs identified by prenatal CMA, including 20 consistent CNVs and 4 inconsistent CNVs. Overall, the DR of cell-free DNA test for pathogenic CNVs ＞2 Mb was 69%. Microdeletions or microduplications at 22q11.2 were the most common CNVs, with a DR of 4/5 (80%) and 3/4 (75%) respectively. CONCLUSION: Cell-free DNA test exhibited a moderate DR for pathogenic CNVs ＞2 Mb among fetuses with ultrasound abnormalities. Cell-free DNA test could provide an opportunity for early screening before the appearance of abnormalities on fetal ultrasound, while further clinical data and cost-effectiveness assessment are needed.

Prenatal features of 17q12 microdeletion and microduplication syndromes: A retrospective case series.

OBJECTIVE: To present the experience on prenatal features of 17q12 microdeletion and microduplication syndromes. MATERIALS AND METHODS: Prenatal chromosomal microarray analysis (CMA) were conducted between January 2015 and December 2018 at a single Chinese tertiary medical centre. Information of cases identified with 17q12 microdeletion or microduplication syndromes were retrospectively collected. Foetal ultrasonographic findings were reviewed, and other information about the gestation week at diagnosis, inheritance and pregnancy outcomes were also included. RESULTS: Ten pregnancies with 17q12 microdeletion and 4 with 17q12 microduplication were identified. The copy number variation (CNV) sizes were 1.39-1.94 Mb in the deleted cases and 1.42-1.48 Mb in the duplicated cases, respectively. All the duplicated and deleted regions included HNF1B and LHX1 genes. Most individuals with 17q12 deletion presented kidney anomalies (9/10), with renal hyperechogenicity being the most common finding (7/10). Fetuses with 17q12 duplication presented a wide phenotypic spectrum, including "double bubble" sign, structural anomalies of the heart and growth anomalies. CONCLUSIONS: Our experience further demonstrated the high correlation between 17q12 microdeletion and renal anomalies especially hyperechogenic kidneys. Structural anomalies of the heart were newly identified phenotypes of 17q12 duplication during prenatal period. Besides, growth anomalies and duodenal atresia might be associated with the duplication.

The application of chromosomal microarray analysis to the prenatal diagnosis of isolated mild ventriculomegaly.

OBJECTIVE: To investigate the clinical value of chromosomal microarray analysis (CMA) in the prenatal diagnosis of genetic abnormalities in fetal isolated mild ventriculomegaly. MATERIALS AND METHODS: This retrospective study reviewed 101 fetuses with isolated mild ventriculomegaly who had undergone invasive prenatal diagnosis at our hospital. CMA was performed in all cases to detect chromosomal aneuploidy as well as copy number variations (CNVs) that are too small to be detected by conventional karyotyping. Real time quantitative PCR (qPCR) or multiplex ligation dependent probe amplification (MLPA) was used to confirm all fetal CNVs <400 Kb. RESULTS: Except for three cases of chromosomal aneuploidy, CMA revealed pathogenic copy number variations (CNVs) in 3.0% (3/101) of the fetuses; these cases demonstrated involvement in the chromosomal regions 15q11.2, 1q21.1 and Xq27.3q28. Furthermore, we detected three likely pathogenic (3.0%) and two variants of uncertain significance (2.0%) among 101 fetuses diagnosed as isolated mild ventriculomegaly on ultrasound examination. CONCLUSION: Our study suggests that CNVs could aid in the risk assessment and genetic counseling in fetuses with isolated ventriculomegaly.

[Mutation analysis and prenatal diagnosis of families affected with Duchenne and Becker muscular dystrophy].

OBJECTIVE: To detect potential mutations for probands from families affected with Duchenne/Becker muscular dystrophy (DMD/BMD), and to carry out prenatal diagnosis through identification of female carriers. METHODS: A total of 43 DMD/BMD families were recruited. Multiplex PCR was used to analyze 18 exons within hotspots for DMD gene deletions. Multiplex ligation-dependent probe amplification (MLPA) was used to detect potential deletions and duplications of DMD gene for 43 patients and 36 females from 32 families. Prenatal diagnosis was performed for 27 families. RESULTS: Deletional mutations were detected in 26 patients with multiplex PCR. In addition, MLPA has detected 3 deletions and 6 duplicational mutations, and the ranges of mutations were all determined. Among 36 female members, 18 were determined as carriers of deletional mutations, 10 were excluded as mutation carriers. The status of remaining 8 could not be determined. For prenatal diagnosis, 3 out of 18 male fetuses were diagnosed as patients and 1 female fetus was identified as carrier. CONCLUSION: MLPA is an accurate and reliable method for detecting deletional/duplicational mutations of DMD gene as well as for prenatal diagnosis and detection of female carriers.

[Molecular genetic study of MECP2 gene for a patient with typical Rett syndrome].

OBJECTIVE: To provide genetic diagnosis and counseling for a 2-year-old girl with typical Rett syndrome through analyzing the methyl-CpG binding protein 2 (MECP2) gene. METHODS: Potential mutation of the MECP2 gene was screened by DNA sequencing and multiplex ligation-dependent probe amplification (MLPA) analysis of members of the family as well as normal controls. Lymphocyte culture for karyotype analysis was carried out for the patient to exclude chromosomal abnormalities. RESULTS: The karyotype of the girl was normal. No variation of the MECP2 gene was detected in the patient by direct sequencing. A heterozygosis variation, c.1072G>A in exon 4 of the MECP2 gene was detected in a normal female control, which was not found in other controls. The son and daughter of the female control were respectively heterozygous and homozygous carriers of the same mutation. By MLPA analysis, a heterozygosis deletion of exon 3 and part of exon 4 was detected in the patient. cDNA amplification and sequencing confirmed the presence of a 1176 bp deletion (c.27-1202del1176). The same deletion was not detected in the parents. CONCLUSION: A large deletion in MECP2 gene was detected with MLPA in a patient featuring typical Rett syndrome. The same deletion was missed by sequencing analysis. With cDNA sequencing, the breakage point of the mutation can be mapped precisely.

Mutation analysis and prenatal diagnosis of EXT1 gene mutations in Chinese patients with multiple osteochondromas.

BACKGROUND: Multiple osteochondromas (MO), an inherited autosomal dominant disorder, is characterized by the presence of multiple exostoses on the long bones. MO is caused by mutations in the EXT1 or EXT2 genes which encode glycosyltransferases implicated in heparin sulfate biosynthesis. METHODS: In this study, efforts were made to identify the underlying disease-causing mutations in patients from two MO families in China. RESULTS: Two novel EXT1 gene mutations were identified and no mutation was found in EXT2 gene. The mutation c.497T > A in exon 1 of the EXT1 gene was cosegregated with the disease phenotype in family 1 and formed a stop codon at amino acid site 166. The fetus of the proband was diagnosed negative. In family 2, the mutation c.1430-1431delCC in exon 6 of the EXT1 gene would cause frameshift and introduce a premature stop codon after the reading frame being open for 42 amino acids. The fetus of this family inherited this mutation from the father. CONCLUSIONS: Mutation analysis of two MO families in this study demonstrates its further application in MO genetic counseling and prenatal diagnosis.

[Mutation analysis and prenatal diagnosis of sporadic DMD/BMD families].

OBJECTIVE: To analyze the pathogenic mutation of sporadic patients in Duchenne/ Becker muscular dystrophy (DMD/BMD) families and to perform prenatal diagnosis, identify the female carriers and evaluate the ratio of de novo mutation in these pedigrees. METHODS: A total of 30 sporadic DMD/BMD families were included. Traditional multiplex PCR was used to detect the 18 exons in the deletion hot area of DMD gene. MLPA was used to detect the deletion or duplication mutations of DMD gene for 30 patients and 28 females from 23 families. Prenatal diagnosis was performed in 19 families. RESULTS: Deletion mutation was detected in 19 patients through mPCR. Twenty-one deletions and 3 duplication mutations were detected by MLPA. Among 21 mothers, 10 mothers were carriers of deletion mutation and two duplication mutation carriers. The other 9 mothers were non-carriers, the mutations in these families were de novo and the ratio was 37.5%. Among seven sisters, five were carriers and two non-carriers. In the prenatal diagnosis families, 2 of 8 female fetuses were carriers and 5 of 12 male fetus patients. CONCLUSION: The MLPA technique has proved to be an accurate and reliable tool not only for the deletion and duplication mutations of DMD patients, but also for the prenatal diagnosis and the female carriers in these families. Prenatal diagnosis;

[Mutational analysis in a family with X-linked spondyloepiphyseal dysplasia tarda].

OBJECTIVE: To detect the mutation of the SEDL gene in an X-linked spondyloepiphyseal dysplasia tarda (SEDL) family. METHODS: Two patients and three females of the X-SEDL family were detected using reverse transcriptase PCR (RT-PCR) and sequence analysis. RESULTS: A G209A mutation of SEDL gene was detected in the cDNA sequences of the patients, which was confirmed by sequence analysis of the exon 4 of the SEDL gene. The daughter of the proband was a carrier of the mutation. CONCLUSION: Since the SEDL gene is relatively small, sequence analysis of cDNA of the SEDL gene was possible after extraction of total RNA followed by RT-PCR. Mutations in the open reading frame can be detected y by cDNA sequencing. It was relatively more rapid and direct than amplifying and detecting the exons one by one.

[Multiplex quantitative PCR detection for female carrier in an X-linked ichthyosis family].

OBJECTIVE: To analyze the pathogenic mutation of an X-linked ichthyosis (XLI) family, and identify the genetic diagnosis of three probable female carriers in this family. To evaluate the availability of different detect methods for steroid sulfatase (STS) gene mutation. METHODS: Peripheral blood samples were collected from the family, including the proband, proband's mother, younger sister, and younger female cousin, and 10 males and 10 females as controls. Ordinary PCR was used to detect whether there was STS gene deletion in the male proband. Then, multiplex quantitative fluorescent PCR (QF-PCR) was used to detect the STS gene in the proband and his 3 female family members. Fluorescence in situ hybridization (FISH) was used to authenticate the results of multiplex QF-PCR method. RESULTS: No amplified product of the exons 1-10 of STS gene deletion was detected by ordinary PCR in the proband. The proband's mother was diagnosed as a carrier, but his sister and cousin were diagnosed as normal females by multiplex QF-PCR. FISH confirmed the results of multiplex QF-PCR. CONCLUSION: Both multiplex QF-PCR and FISH are effective to detect the complete deletion mutation of STS gene and identify the female carrier, and multiplex QF-PCR is more convenient and automatic compared with FISH.

[Prenatal diagnosis of oculocutaneous albinism type II and discovery of two novel mutations].

OBJECTIVE: To investigate the genotype of oculocutaneous albinism type II (OCA2) and perform prenatal gene diagnosis for OCA2. METHODS: Peripheral blood samples were collected from a 9-year-old girl with OCA and her parents, the mother being pregnant. PCR, automatic sequence analysis and denaturing high performance liquid chromatography (DHPLC) were used to analyze the TYR gene and P gene so as to screen the OCA genes. Amniocentesis was conducted when the mother was 20-week pregnant and the amniotic cells underwent screening of the 2 specific mutations. Peripheral blood samples were collected from 100 healthy persons without phenotype of OCA to undergo genetic analysis. RESULTS: The TYR gene of the proband did not show any mutation, and 2 new mutations were found in her P gene: p. N476D (c. 1426 A>G) and p.Y827H (c.2479T>C). Her father and mother were heterozygote of Y827H and N476D respectively. Based on these findings the amniotic cells underwent sequencing of enlarged fragments of the exons 14 and 24 containing the mutation sites. The result showed that the fetus only got the maternal N476D mutation and didn't get the paternal Y827H mutation. So the fetus was predicted to be a carrier of OCA2 with normal appearance. The baby was born normal later as predicted. None of these 2 mutations was found in the 100 healthy persons. CONCLUSION: This is a success of prenatal gene diagnosis of OCA2. Two novel mutations of the P gene related to OCA have been discovered.

[A novel P gene mutation in a Chinese family with oculocutaneous albinism].

OBJECTIVE: To investigate gene mutations of a consanguineous family with two oculocutaneous albinism (OCA) patients. METHODS: Genomic DNA was prepared from peripheral leukocytes. All of the exons and flanking introns of P gene and TYR gene were PCR-direct-sequenced. Hha I restriction fragment length polymorphism in codon 787 of the P gene was studied in the family and 102 unrelated normal Chinese individuals. RESULTS: Although no mutations were found in TYR gene, a missense mutation A787T was found in P gene. Two patients of the family were both homozygous for A787T. Their parents and brother were heterozygous for the mutation. The mutation was not observed among 102 normally pigmented subjects. CONCLUSION: The A787T mutation is not a common polymorphism among normal Chinese and it seems most likely to be a pathological OCA2 mutation. This is the first report on the study of gene diagnosis in Chinese OCA2 patients.

[A new form of Oculocutaneous albinism, OCA4].

Oculocutaneous albinism (OCA) is a complex genetic disease with great clinical heterogeneity. Four different types of OCA have been reported to date (OCA1, OCA2, OCA3, and OCA4). OCA4 was firstly reported in a Turkish OCA patient. The gene responsible for OCA4 is the human homologue of the mouse underwhite (uw) gene, which encodes the mem-brane-associated transporter protein (MATP). MATP gene is located on chromosome 5p13.3 and is divided into 7 exons and 6 introns. MATP gene is transcriptionally modulated by MITF, and encodes a protein of 530 amino acids. There are at least 18 pathologic mutations and 8 non-pathologic polymorphisms have been found.

[Prenatal gene diagnosis of oculocutaneous albinism type I].

OBJECTIVE: Mutation analysis and prenatal gene diagnosis for the mutated tyrosinase (TYR) gene in two families with oculocutaneous albinism type I (OCA1). METHODS: To define the fetus genotypes and gene mutation sites, the PCR and sequencing techniques were applied to amplify and analyze the regions of exon, exon-intron and promoter of TYR gene in probands and their parents of 2 families. RESULTS: The patient or proband of family 1 showed as a compound heterozygote with mutants R278X and 929insC. However, the fetus did not get any one of the two mutations, and so was with a normal genotype and phenotype. The parents of proband in family 2 were heterozygous with IVS4+ 3A>T or G253E respectively, but their fetus was heterozygous only with IVS4+3A>T but without G253E, and so was a carrier as his father. CONCLUSION: In the mainland of China, the prenatal gene diagnosis of OCA1 is reported for the first time.

[Mutations and polymorphisms of the P gene associated with oculocutaneous albinism type II].

Oculocutaneous albinism typeII(OCA2), the most common type of albinism, is an autosomal recessive disorder. It is caused by mutations in the P gene, which is located on chromosome 15q11.1-q12 and divided into 24 exons and 23 introns. P gene codes for 838-amino-acid integral membrane protein with 12 putative transmembrane domains, but the exact function is not clear yet. There are at least 60 pathologic mutations and 43 non-pathologic polymorphisms have been found. Pathologic mutations include missense mutations, nonsense mutations, frameshift mutations and splice-sit mutations. But unlike TYR gene, most of P gene mutations are located on the C-terminal and don' t cluster in defined regions. It is difficult to define the pathologic mutations since many non-pathologic polymorphisms also lie in exons. Some non-pathologic missense mutations may be associated with phenotypic variation in normally pigmented individuals and need to further study.