Prenatal diagnosis of Chinese families with phenylketonuria.

The aim of this study is to investigate the ability to prenatally diagnose phenylketonuria (PKU) by using phenylalanine hydroxylase (PAH) gene mutation analysis combined with short tandem repeat (STR) linkage analysis in 118 fetuses from 112 Chinese families. Genomic DNA was extracted from the peripheral blood from members of 112 families and the exons and exon-intron boundaries of the PAH gene were amplified by PCR. PCR products were analyzed by bi-directional Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). The three variable number of tandem repeat (VNTR) markers PAH-1, PAH-26, PAH-32 were used in the prenatal diagnosis for the PKU families. We identified a spectrum of 63 different mutations, including 61 point mutations and indels, two large exon deletion mutations, and five novel mutations. A substantial proportion of mutant alleles were accounted for by p.R243Q (15.62%), EX6-96AG (9.82%), p.V399V (7.59%), p.Y356X (6.70%), and p.R413P (5.36%). The same mutations were identified in 31 prenatally genotyped fetuses. We identified 58 fetuses that carried only one mutant allele and 29 fetuses that carried no mutations of PAH and were presumed normal. PAH gene mutation analysis combined with STR linkage analysis can provide rapid and accurate prenatal diagnosis for PKU families.

Mutation analyses and prenatal diagnosis in families of X-linked severe combined immunodeficiency caused by IL2Rγ gene novel mutation.

We investigated the feasibility of interleukin-2 receptor gamma (IL2Rγ) gene based on gene mutation analysis and pre-natal diagnosis of X-linked severe combined immunodeficiency (X-SCID). Blood samples of patients and their parents of X-SCID (family 1) and X-SCID (family 2) were collected. IL2Rγ gene sequences of the 2 families were analyzed using bi-directional direct sequencing by polymerase chain reaction. DNA sequence changes in the IL2Rγ gene exon region and shear zone were also analyzed. We also sequenced the IL2Rγ gene in 100 healthy individuals. Prenatal genetic diagnoses for a high-risk fetus in family 1 were performed by chorionic villus sampling after determining each family's genotypes. The suspect fe-male in family 1 underwent carrier detection. Two novel mutations of IL2Rγ gene were identified, including c.361-363delGAG (p.E121del) in the patient and his mother in family 1, and c.510-511insGAACT (p.W173X) heterozygous mutation in the proband's mother in family 2. These mutations were absent in the 100 controls. Prenatal diagnosis of early pregnancy in the female fetus of family 1 was performed; the fetus was heterozygous, which was confirmed at postnatal follow-up. The suspect female in family 1 showed no mutation in carrier detection. The novel p.E121del and p.W173X mutations in IL2Rγ may have been the primary causes of disease in 2 families with X-SCID. In couples with an X-SCID reproductive history, prenatal gene mutation analysis of IL2Rγ can effectively prevent the birth of children with X-SCID and carrier detection for suspected females.

A novel 3-base pair deletion of the CRYAA gene identified in a large Chinese pedigree featuring autosomal dominant congenital perinuclear cataract.

Congenital cataract is caused by reduced transparency of the lens resulting from metabolic disorders during the fetal period. The disease shows great heterogeneity both clinically and genetically. We identified a 4-generation ethnic Han Chinese family affected by autosomal dominant congenital perinuclear cataract. The patients underwent full clinical and ophthalmologic examinations to rule out any concomitant disorders. Blood samples were collected and genomic DNA was extracted. Potential mutations in the candidate gene alpha A crystallin (CRYAA) were screened. Prenatal diagnosis was then provided for a fetus of the affected proband by chorionic villus sampling. In all patients, DNA sequencing of the CRYAA gene revealed a novel 3-bp deletion mutation in exon 3 (c.246_248delCGC), which led to deletion of codon 117 encoding arginine (p.117delR) in the peptide chain. The same mutation was not found among unaffected and healthy individuals. Bioinformatic analysis revealed that although the c.246_248delCGC is an 'in-frame' mutation, removal of arginine resulted in a significant change in the protein structure. The fetus did not possess this mutation and was confirmed to be healthy at 1-year follow-up. A novel disease-causing mutation, c.246_248delCGC (p.117delR), of the CRYAA gene has been identified in a Chinese family with autosomal-type perinuclear congenital cataracts. This is also the first report of prenatal diagnosis of this type of congenital cataract.

Analysis and application of ATP7B gene mutations in 35 patients with hepatolenticular degeneration.

We investigated the genetic mutations involved in Wilson's disease to improve prenatal genetic diagnosis and presymptomatic diagnosis. The polymerase chain reaction (PCR) was used to amplify the exons and exon-intron boundaries of the ATP7B gene in 35 Wilson's disease pedigrees. The PCR products were further analyzed by Sanger sequencing. Prenatal genetic diagnoses were performed by chorionic villus sampling after the genotypes of parents of the probands were identified. The overall mutation detection frequency was 92.9%. A total of 24 distinct mutations were detected, seven of which are novel: A1291T (c.3871G>A), c.2593_2594insGTCA, c.2790_2792delCAT, c.3661_3663delGGG, c.3700delG, c.4094_4097delCTGT, and IVS6+1G>A. Three mutations, R778L (c.2333G>T) (45.7%), A874V (c.2621C>T) (7.1%), and P992L (c.2975C>T) (7.1%) are relatively frequent. Two presymptomatic patients were detected through familial screening, and they began taking medicine after diagnosis. Of the subjects with Wilson's disease pedigrees who had received a prenatal genetic diagnosis, three fetuses were normal and one was a carrier. Twenty-four distinct mutations were identified, and our knowledge of the population genetics of Wilson's disease in China has therefore improved. For pedigrees with the Wilson's disease, genetic counseling, prenatal diagnosis, and presymptomatic diagnosis by Sanger sequencing and haplotype analysis are feasible.

Mutation analysis and prenatal diagnosis for three families affected by isolated methylmalonic aciduria.

Isolated methylmalonic acidemia (MMA) is a genetically heterogeneous disorder caused mainly by deficiency of methylmalonyl-CoA mutase. In the present study, we analyzed MUT gene mutations in 3 Chinese couples with a birth history of isolated MMA. We also provided prenatal diagnoses for the detected mutation. Exons and exon-intron boundaries of the MUT gene were analyzed by polymerase chain reaction and direct sequencing. Prenatal genetic diagnoses were performed by chorionic villus sampling after the genotypes of parents were determined. Six heterozygous mutations in the MUT gene were identified in the 3 families, including c.1880A>G (p.H627R) and IVS9-1G>A for family 1, c.1741C>T (p.R581X) and c.729insTT (p.D244fX39) for family 2, and c.616C>T (p.Q206X) and c.1280G>A (p.G427D) for family 3. Among these, c.616C>T (p.Q206X), c.1280G>A (p.G427D), IVS9-1G>A, and c.1741C>T (p.R581X) were novel mutations. These mutations were not detected in 100 normal controls. The fetus in pedigree 3 was free of the mutations carried by the parents, while the fetuses in pedigrees 1 and 2 were heterozygous mutation carriers. All 3 families decided to continue with their pregnancies and the neonates did not show any symptoms of MMA after birth. Our results indicated that mutations in the MUT gene are the primary cause of isolated MMA, and that most mutations were novel. For families with early-onset isolated MMA, direct sequencing of the MUT gene is crucial for genetic counseling, prenatal diagnosis, and identification of carriers.

Bioinformatics analysis of biomarkers and transcriptional factor motifs in Down syndrome

In this study, biomarkers and transcriptional factor motifs were identified in order to investigate the etiology and phenotypic severity of Down syndrome. GSE 1281, GSE 1611, and GSE 5390 were downloaded from the gene expression ominibus (GEO). A robust multiarray analysis (RMA) algorithm was applied to detect differentially expressed genes (DEGs). In order to screen for biological pathways and to interrogate the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database, the database for annotation, visualization, and integrated discovery (DAVID) was used to carry out a gene ontology (GO) function enrichment for DEGs. Finally, a transcriptional regulatory network was constructed, and a hypergeometric distribution test was applied to select for significantly enriched transcriptional factor motifs. CBR1, DYRK1A, HMGN1, ITSN1, RCAN1, SON, TMEM50B, and TTC3 were each up-regulated two-fold in Down syndrome samples compared to normal samples; of these, SON and TTC3 were newly reported. CBR1, DYRK1A, HMGN1, ITSN1, RCAN1, SON, TMEM50B, and TTC3 were located on human chromosome 21 (mouse chromosome 16). The DEGs were significantly enriched in macromolecular complex subunit organization and focal adhesion pathways. Eleven significantly enriched transcription factor motifs (PAX5, EGR1, XBP1, SREBP1, OLF1, MZF1, NFY, NFKAPPAB, MYCMAX, NFE2, and RP58) were identified. The DEGs and transcription factor motifs identified in our study provide biomarkers for the understanding of Down syndrome pathogenesis and progression.

Bioinformatics analysis of biomarkers and transcriptional factor motifs in Down syndrome.

In this study, biomarkers and transcriptional factor motifs were identified in order to investigate the etiology and phenotypic severity of Down syndrome. GSE 1281, GSE 1611, and GSE 5390 were downloaded from the gene expression ominibus (GEO). A robust multiarray analysis (RMA) algorithm was applied to detect differentially expressed genes (DEGs). In order to screen for biological pathways and to interrogate the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database, the database for annotation, visualization, and integrated discovery (DAVID) was used to carry out a gene ontology (GO) function enrichment for DEGs. Finally, a transcriptional regulatory network was constructed, and a hypergeometric distribution test was applied to select for significantly enriched transcriptional factor motifs. CBR1, DYRK1A, HMGN1, ITSN1, RCAN1, SON, TMEM50B, and TTC3 were each up-regulated two-fold in Down syndrome samples compared to normal samples; of these, SON and TTC3 were newly reported. CBR1, DYRK1A, HMGN1, ITSN1, RCAN1, SON, TMEM50B, and TTC3 were located on human chromosome 21 (mouse chromosome 16). The DEGs were significantly enriched in macromolecular complex subunit organization and focal adhesion pathways. Eleven significantly enriched transcription factor motifs (PAX5, EGR1, XBP1, SREBP1, OLF1, MZF1, NFY, NFKAPPAB, MYCMAX, NFE2, and RP58) were identified. The DEGs and transcription factor motifs identified in our study provide biomarkers for the understanding of Down syndrome pathogenesis and progression.