Objective: To analyse the genetic cause of a proband with mitochondrial disease caused by FASTKD2 gene variation and uniparental disomy. Methods: Detailed medical history of a child suspected "mitochondrial disease" were inquired in Peking University First Hospital on November 23, 2017. c.810_820dup homozygous variation in FASTKD2 gene was found by high-throughput sequencing, and her mother had heterozygous variation, but her father didn't have such variation, which didn't conform to the genetic law of variation. Further clinical examinations and molecular genetic tests were carried out. The venous blood of the child and her parents was drawn, and genomic DNA was extracted. Sanger sequencing, polymerase chain reaction (PCR) testing, short tandem repeat (STR) analysis, chromosome microarray analysis and loss of heterozygosity (LOH) genetic relationship analysis were performed on the proband and the parents to determine the variation. Results: The clinical manifestations, physical examination and laboratory examination of the child supported the diagnosis of mitochondrial disease. c.810_820dup(p.Ser274Phefs*8) homozygous variant in FASTKD2 gene was identified. Sanger sequencing indicated that the mother was a heterozygote of the variant, while the father had no such variation, which did not conform to the genetic law. PCR testing and Sanger sequencing review to eliminate sampling errors, PCR amplification and sequencing errors. Non-biological father was excluded by STR analysis. Three large segmental LOH of FASTKD2 gene were found by chromosome microarray analysis, then the LOH relative analysis verified the child was a mixed maternal uniparental disomy of chromosome 2. The child was diagnosed as mitochondrial disease caused by oxidative phosphorylation coupling defect of type 44. Conclusions: In this study, an autosomal recessive mitochondrial disease which does not conform to the genetic law was found, and it was confirmed that this mitochondrial disease family had both pathogenic variation and uniparental disomy phenomenon. It was diagnosed as mitochondrial disease caused by type 44 oxidative phosphorylation coupling defect.

The four alien farmland weeds of genus Veronica (i.e. V. arvensis, V. didyma, V. hederifolia and V. persica) have successfully colonized in China, but caused different ecological consequences in the colonized habitats. However, the key biological traits conferring bioinvasion differences under different light conditions among the four alien species of Veronica remain unknown. A comprehensive contrastive analysis experiment was conducted to assess the contribution of the intensity of photosynthetic and sexual and asexual reproductive traits of the four alien Veronica weeds to their invasion level in both field trial and laboratory. The field survey showed that V. persica had the highest invasion level, followed by V. didyma, V. hederifolia and V. arvensis. Their invasiveness was mainly attributed to photosynthetic-related parameters (LMA) and asexual reproduction traits (the ratio of adventitious roots) out of all the 22 tested indexes. The photosynthetic-related and some asexual reproduction indexes from separate determinations under both sun and shade conditions showed that V. persica was able to adapt to strong illumination but was more tolerant of shade than the other species. This adaptive differentiation to illumination conferred different competitiveness over crops on the four alien Veronica weeds by allocating resources to the biomass of each organ in farmland. It may be concluded that the adaptability to illumination conditions and the asexual reproduction traits may endow their successful invasion and become different important farmland weeds.

AbstractBackgroundVariants in the ATP binding cassette protein subfamily D member 1 (ABCD1) gene are known to cause X‐linked adrenoleukodystrophy (X‐ALD). This study focused on the characteristics of ABCD1 variants in Chinese X‐ALD families and elucidated the value of genetic approaches for X‐ALD.Methods68 male probands diagnosed as X‐ALD were screened for ABCD1 variants by the Sanger sequencing of polymerase chain reaction (PCR) products and multiplex ligation‐dependent probe amplification (MLPA) combined with long‐range PCR. Prenatal diagnosis was performed in 20 foetuses of 17 probands’ mothers. Descriptive statistics were used to summarise the gene variants and prenatal diagnosis characteristics and outcomes.ResultsThis study allowed the identification of 61 variants occurring in 68 families, including 58 single nucleotide variants or small deletion/insertion variants and 3 large deletions. Three probands with no variants detected by next‐generation sequencing were found to have variants by PCR‐sequencing. Prenatal diagnosis found that 10 of the 20 foetuses had no variants in ABCD1.ConclusionPCR primers that do not amplify the pseudogenes must be used for PCR‐sequencing. MLPA combined with long‐range PCR can detect large deletions and insertions, which are usually undetectable by PCR‐sequencing. Prenatal diagnosis could help to prevent the birth of infants with X‐ALD.

Objective:To explore the role of parental origin verification in chromosomal microarray analysis (CMA) on the determination of the clinical significance of copy number variations (CNVs).Methods:This retrospective study collected clinical information from 73 core families who underwent prenatal diagnosis at Peking University First Hospital from November 2017 to December 2019. Indications for prenatal diagnosis included ultrasound abnormality in 54 cases (including 12 with thickened nuchal translucency (≥2.5 mm), four with fetal growth restriction, seven with abnormal pregnancy history, and 31 with isolated ultrasound abnormality), NIPT indicated high-risk in four cases, advanced age in nine cases, abnormal pregnancy history alone in three cases, intrauterine death in two cases and one with maternal mental retardation. Genomic DNA of amniotic fluid sample, chorionic villi, cord blood, fetal tissues, and fetal heart blood were extracted using genomic DNA extraction kit. The CNVs of prenatal samples in 73 subjects were analyzed using array-based comparative genomic hybridization (array-CGH) analysis and single nucleotide polymorphism array (SNP-array). Peripheral blood DNA of the couples, and relevant families if necessary, were collected and analyzed in the same way. The results of parental origin detection in CMA were summarized.Results:A total of 76 CNVs were detected in these 73 samples, out of which nine were pathogenic and parental origin detection revealed that six were de novo, two were maternally, and one was paternally inherited; six CNVs were likely pathogenic, including three de novo, two maternally inherited and one paternally inherited; 20 CNVs were variants of uncertain significance, including five paternally inherited, three maternally inherited and 12 de novo; 41 CNVs were likely benign, among which 38 were inherited from parents with normal phenotype. Conclusions:Parental origin verification plays an important role in explaining the clinical significance of detected fetal CNVs and thereby can help to analyze its clinical effect and reproductive risk.

Objective To determine changes in mitochondrial DNA (mtDNA) copy number in peripheral blood in Rett syndrome caused by methyl-CpG-binding protein-2 (MECP2) variants and explore the mechanism of mitochondrial dysfunction in Rett syndrome. Study design Female patients who were diagnosed with Rett syndrome and had an MECP2 variant (n = 142) were recruited in this study, along with the same number of age- and sex-matched healthy controls. MtDNA copy number was quantified by real-time quantitative polymerase chain reaction with TagMan probes. The differences in mtDNA copy number between the Rett syndrome group and the control group were analyzed using the independent-samples t test. Linear regression, biserial correlation analysis, and one-way ANOVA were applied for the correlations between mtDNA copy number and age, clinical severity, variant types, functional domains, and hot-spot variants. Results MtDNA copy number was found to be significantly increased in the patients with Rett syndrome with MECP2 gene variants compared with the control subjects. Age, clinical severity, variant types, functional domains, and hot-spot variants were not related to mtDNA copy number in patients with Rett syndrome. Conclusions MtDNA copy number is increased significantly in patients with Rett syndrome, suggesting that changes in mitochondrial function in Rett syndrome trigger a compensatory increase in mtDNA copy number and providing new possibilities for treating Rett syndrome, such as mitochondria-targeted therapies.

Objective:To summarize the characteristics of genetic variation and prenatal diagnosis in pedigrees with X-linked adrenoleukodystrophy (X-ALD) and elucidate the value of prenatal diagnosis in preventing the birth of children with X-ALD.Methods:Twenty pedigrees, clinically diagnosed with X-ALD in Peking University First Hospital from November 2012 and March 2019, were included in this retrospective study. Genomic DNA was extracted from peripheral blood and amniotic fluid or chorionic villi samples of probands and their families for detecting variants in ATP-binding cassette subfamily D member 1 ( ABCD1) gene using polymerase chain reaction (PCR)-Sanger sequencing. Linkage analysis was also performed on five microsatellite markers near ABCD1 gene to exclude maternal contamination. Characteristics of ABCD1 gene variants and prenatal diagnosis of X-ALD pedigrees were summarized by descriptive statistics. Results:Twenty ABCD1 gene variants were identified in the 20 pedigrees. The variants in three probands that were not detected by next-generation sequencing were identified by PCR-Sanger sequencing. Among the mothers of the 20 probands, 17 carried ABCD1 variants and three did not. We performed 24 prenatal diagnoses on 20 pregnancies (24 fetuses) and identified eight fetuses with variants who were finally terminated. The 16 cases without variants were born alive. The validation results obtained after termination or delivery were consistent with those performed prenatally. Conclusions:No hotspot variants in ABCD1 gene are detected in these X-ALD patients and most variants are maternally inherited. PCR-Sanger sequencing is an effective method for detecting ABCD1 variants. Prenatal diagnosis for mothers who had a body with X-ALD could prevent another one from birth.

Objective:To study the value of chromosome microarray analysis (CMA) application in children with developmental delay (DD), intellectual disability (ID), autistic spectrum disorder (ASD) and multiple congenital anomalies (MCA).Methods:Genomic DNA was extracted from peripheral blood samples. Array-based comparative genomic hybridization (array-CGH) analysis and single nucleotide polymorphism array (SNP-array) were performed in 1 320 children with DD/ID, ASD, with or without epilepsy and MCA who were admitted to Peking University First Hospital from 2014 to 2019. The results of genetic etiology test of CMA in children with mental retardation or global DD was summarized.Results:Of 1 320 samples, there were 10 cases of aneuploid abnormality, 6 cases of uniparental disomy and one case of mosaicism, respectively. Pathogenic copy number variations (CNVs) were found in 320 cases and pathogenic CNVs were detected in 23 cases, with a combined detection rate of 26% (343/1 320). CNVs of uncertain clinical significance occurred in 107 cases, accounting for 8.1% (107/1 320). There were 25 cases of possible benign CNVs, accounting for 2% (25/1 320), while benign CNVs were reported in 20 cases, accounting for 1.5% (20/1 320). The detection rate of MCA with DD/ID was 39.8% (130/327).Conclusions:CMA has the advantages of high resolution and covering the whole genome. It can detect the chromosomal abnormalities, microdeletions and duplications seen under the microscope, thus the genetic etiology of children with mental retardation or global DD can be diagnosed.

To the Editor: Phenylketonuria (PKU) is an autosomal recessive genetic disease caused by pathogenic variants in the phenylalanine hydroxylase (PAH) gene encoding phenylalanine hydroxylase, a key enzyme in the metabolism of phenylalanine. Early low-phenylalanine diet improves most of the neuropsychological disorders, but it is difficult to be maintained for a long period of time.[1] To date, 1184 variants in PAH gene, including missense, splicing, nonsense, insertion and deletion variants, have been identified. The distribution of the variants is quite variable in ethnic groups. Genetic testing and prenatal diagnosis are effective to prevent PKU families from transmitting the pathogenic PAH alleles to their progeny. However, only a few reports about the prenatal diagnosis of PKU from north China have been found in the literature. Here we summarized the results of variant detection in 157 probands and their parents, and prenatal diagnosis of 103 fetuses from 95 PKU families. This study was approved by the Research Ethics Committee of Peking University First Hospital. Informed consent was obtained from the probands or their guardians and their family members. A total of 157 probands with their parents were examined for the variants in PAH gene during the period from May 2012 to December 2018. The age of the probands ranged from 1 month to 17 years, the male to female ratio was 1:0.92. Most of them lived in north China. All the probands had higher levels of plasma phenylalanine (>2 mg/dL), and the diagnosis of tetrahydrobiopterin (BH4) deficiency was excluded by a BH4-loading test. Genomic deoxyribonucleic acid (DNA) was isolated from peripheral lymphocytes of the probands and their parents by a QuickGene DNA Whole Blood Kit (KURABO, Osaka, Japan). The 13 exons and their flanking sequences of the probands were amplified by polymerase chain reaction (PCR) that contained 50 ng DNA, 2.5 mmol/L each deoxy-ribonucleoside triphosphates (dNTPs) 2 μL, 10× reaction buffer 5 μL, 10 μmol/L each primers 1 μL, and 2.5 units of Taq DNA polymerase in a total volume of 50 μL. PCR products were purified and sequenced in an ABI 3130XL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Sequencing results were compared with the transcript (NM_000277) of PAH gene and its genomic sequence (GRCh38/hg38). Detected variants were further searched in the three databases PAHvdb (www.biopku.org/pah/), ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/), and HGMD (http://www.hgmd.cf.ac.uk/ac/). A novel variant not found in these databases was evaluated by the online predictive tools of sorting tolerant from intolerant (SIFT) (http://provean.jcvi.org/index.php), PROVEAN (http://provean.jcvi.org/index.php), and PolyPhen2 (http://genetics.bwh.harvard.edu/pph2/) to predict pathogenic effect of the mutant protein. Variants found in the probands were then examined in their respective father and mother. For probands without pathogenic variants or only one pathogenic variant found, the DNA samples were subjected to multiplex ligation-dependent probe amplification (MLPA; MLPA P055 kit, MRC-Holland, Amsterdam, Netherlands) to detect large insertions, deletions, or duplications in PAH gene. MLPA products were separated in ABI 3130XL Genetic Analyzer and analyzed by Coffalyser. Net (MRC-Holland). In the 157 families, prenatal diagnosis was performed for 95 pregnant mothers, in which eight mothers were pregnant twice with prenatal diagnosis twice. DNA samples were extracted from chorionic villi, amniotic fluid, or abortion tissues (for verification of affected fetuses after abortion) using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) and subjected to the same PCR-direct sequencing and/or MLPA procedures as described above. In addition, six short tandem repeats (STR) markers nearby PAH were amplified by PCR and separated on an ABI 3130XL Genetic Analyzer. Genotypes of the six STR markers were compared between mother and the fetus to exclude false results due to maternal blood contamination. Among the 157 probands, 145 probands have two pathogenic alleles, including one proband with two pathogenic variants in one allele and one pathogenic variant in another allele, ten probands have only one pathogenic allele, and no pathogenic alleles were found in two probands. There were total 80 kinds of pathogenic variants, including 71 point nucleotide substitutions, seven small insertions/deletions, and two large deletions, resulting in 52 missense variants in 159 alleles, 13 splicing variants in 82 alleles, seven premature terminations in 46 alleles, five frame shifts in six alleles, two large deletions in four alleles, and one amino acid deletion in four alleles. The spectrum of the 301 variants listed in Supplementary Table 1, https://links.lww.com/CM9/A525, in which the variant of 163_164insATAT is a novel variant not stored in the above three databases. The most prevalent variants were R243Q, splicing variant due to c.611A>G and splicing variant due to c.1197A>T, accounting for 17.9% (54/301), 9.0% (27/301), and 8.3% (25/301) of the variant alleles, respectively. The highest frequency of exon and its flanking sequences in which pathogenic variants (excluding large deletions/duplications) locate was exon 7, followed by exon 11, exon 6, exon 12, and exon 3. MLPA was performed in 13 probands and found one large deletion of exon 1 and its upstream region in two probands and one large deletion of exon 4/exon 5 in the other two probands. Prenatal diagnosis of PKU was performed in 103 fetuses in 95 of the 157 families. Thirty fetuses (29.1%, 30/103) were identified as PKU (carrying two pathogenic variants); all of the families chose abortion, and the pathogenic variants were confirmed by testing the abortion tissues. Fifty-two (50.5%, 52/103) fetuses were identified as PKU carriers (carrying one pathogenic variant), and 21 (20.4%, 21/103) as normal fetuses (no pathogenic variant found). Most of the carrier fetuses and normal fetuses were born (a few of them were aborted by other reasons), and their genotypes of PAH gene were confirmed by testing peripheral blood after birth. Figure 1 showed the importance of molecular diagnosis and prenatal diagnosis for PKU in a family as an example. After the molecular diagnosis of PKU in the proband (III1) and her parents (II1, II2), the proband's aunt (II4) and uncle (II3) were also at the risk to have a PKU baby. Molecular diagnosis revealed that both II3 and II4 were the carriers of PKU. Genetic counseling was then provided to prevent the two pairs of couples from delivery of another PKU case.Figure 1: Pedigree of a phenylketonuria family. After the molecular diagnosis of PKU in the proband (III1) and her parents (II1, II2), the proband's aunt (II4) and uncle (II3) were also at the risk to have a PKU baby. Molecular diagnosis revealed that both II3 and II4 were the carriers of PKU.PAH gene locates in human chromosome 12q23.2, consisting of 13 exons that encode a polypeptide of 452 amino acid residues. Mutant phenylalanine hydroxylase blocks the metabolism of phenylalanine to tyrosine. The accumulation of phenylalanine leads to the alterations of cerebral myelination and protein synthesis and reduced levels of serotonin, dopamine, and noradrenaline in the brain.[2] Eventually, severe mental retardation and neurobehavioral abnormalities are present in these children. Neonatal screening for PKU is only useful for the early treatment of PKU. During the period from 2014 to 2017 in the Haidian District of Beijing city, screened for PKU among 176,340 newborns, in which 33 newborns were confirmed to have PKU with the incidence of 1/5344.[3] In this cohort of PKU probands, the most prevalent variants of R243Q, splicing variants of c.611A>G and c.1197A>T accounted for 35.2% (106/301) of the variants, similar to the reports from other regions in China and Korea.[4,5] In contrast in Japan, the most prevalent variant was R413P.[6] The R243Q variant causes a mutant phenylalanine hydroxylase which has only <10% normal activity in the eukaryotic cell expression system.[7] The novel variant of 163_164insATAT we found causes frameshift and premature termination of the polypeptide, which is a definite pathogenic variant. Two large deletions, exon 1 and its upstream region in two cases and exon 4/exon 5 in other two cases, were identified by MLPA in the 13 probands in which two variant alleles were not found by PCR-Sanger sequencing. Chen et al[8] reported that three large deletion alleles (exon 1 and its upstream region, exon 4/exon 5, and exon 5) were disclosed in 17 PKU families without two pathogenic variants. Yan et al[9] examined 43 PKU patients with none or only one variant allele by MLPA and identified that 22 PKU patients had 24 (51.1%) large deletion/duplication alleles, of which Ex1del3758 was detected in ten cases and Ex4_5del in four cases, similar to our findings. Therefore, the large deletions of exon 1 and exon 4/exon 5 may be relatively common in Chinese PKU patients. No variant hotspot in PAH gene exists in this cohort of PKU patients. The variants were distributed in all 13 exons. The highest frequency of exon and its flanking sequences in which variants locate was exon 7, followed by exon 11, exon 6, exon 12, and exon 3. Zhang et al[10] examined the variants in exons 3, 5, 6, 7, 10, 11, and 12 of PAH gene in 40 PKU families and demonstrated that most variants concentrated in exon 7, followed by exons 6, 11, and 3, which was similar to our results. Therefore, these exons can be chosen first for variant screening. The next-generation sequencing technology has become a powerful tool for the diagnosis of genetic diseases.[4,11] Regular PCR-Sanger sequencing and MLPA could detect 95.6% (301/314) pathogenic alleles in PAH gene in this cohort of PKU patients, suggesting that the classic methods are still effective for the genetic diagnosis of PKU. In families that both the husband and wife who carrying a pathogenic variant in PAH, the possibility of giving birth of a PKU baby is 25%, theoretically. Prenatal diagnosis is the unique way for PKU families to prevent the birth of infant with PKU case. Technologically, genotyping of several STR markers must be included to prevent misdiagnosis due to maternal blood contamination in fetal samples.[12] The six highly polymorphic STR markers that we used for linkage analysis were located around the PAH gene, two upstream, three downstream, and one in intron 3 of PAH gene. In case the fetal samples was contaminated by maternal blood, DNA extracted from cultured amniotic fluid cells or chorionic villi cells must be used to obtain accurate results. Prenatal diagnosis using chorionic villi is usually performed at 11 to 13th week of gestation, and the earlier molecular diagnosis of the fetus is obtained, the less physical and psychological damages to the pregnant woman when the fetus is affected and the pregnancy is terminated. However, abortion due to the manipulation of chorionic villi sampling is relatively high, and the presence of placental chimerism may affect the accuracy of the results. In contrast, amniocentesis is usually performed at 16 to 23rd week of gestation and is relatively safe. However, the later the molecular diagnosis of the fetus, the higher the risk of abortion when the fetus is affected. Here we present the spectrum of variants in PAH gene in PKU patients in north China. No variant hotspot in PAH gene was found. The variants were frequently detected in exon 7. Prenatal diagnosis is the unique way to prevent the progeny of heterozygous couples from PKU. Conflicts of interest None.

Objective:To summarize the prenatal diagnostic characteristics of monogenic global developmental delay/intellectual disability(GDD/ID) pedigrees.Methods:This study retrospectively collected the prenatal molecular diagnostic results of 43 pedigrees that were affected with monogenic GDD/ID in the genetic counseling clinic of Peking University First Hospital from January 2015 to June 2019. The results of prenatal molecular tests were validated after birth or pregnancy termination. Pregnancy outcomes and healthy condition of the offspring were followed up. All data were analyzed by descriptive statistical analysis.Results:Among the 43 pedigrees, 24 were affected with autosomal recessive inheritance (AR) GDD/ID, in which six (25%) fetuses were found to carry two pathogenic variants; 13 (55%) had only one pathogenic variant; five (20%) did not harbor any variant. GDD/ID inherited in an autosomal dominant inheritance (AD) pattern was found in 13 pedigrees, in which 11 fetuses carried no variants while the other two fetuses had the same variants as the proband had (in one pedigree, a low-level variant was detected in the peripheral blood sample of the father while absent in peripheral blood samples of parents in the other pedigree, so it was suspected that the variants of these two affected fetuses were inherited from parental mosaicism). In the other six pedigrees with X-linked inheritance (XL) of GDD/ID, one male fetus was found to harbor the pathogenic variant, while no variants were detected in the others. Maternal contamination was excluded in all prenatal samples using short tandem repeat for linkage analysis. Postnatal validations were consistent with the prenatal tests. All nine affected fetuses were terminated, and the other thirty-four children were delivered and in good health.Conclusions:Prenatal molecular diagnostic test is an effective method to detect pathogenic variants during the first and second trimesters for pedigrees affected by monogenic GDD/ID. For pedigrees affected with AD or XL patterns caused by de novo mutations, potential parental mosaicism should be noted and prenatal diagnostic tests are also recommended.

Objective To analyze the prenatal clinical characteristics and genetic diagnosis of two fetuses with chromosome 17q12 deletion syndrome mainly manifested by renal structural abnormalities. Methods Clinical data of two pregnant women admitted to Peking University First Hospital in 2017 due to ultrasound indication of fetal kidney structure abnormality in the second trimester were collected. Results of fetal chromosome karyotype analysis and array-based comparative genomic hybridization (aCGH), and aCGH detection of peripheral blood in the two couples were reviewed. Results (1) In both pregnancies of case 1 and case 2, no abnormal chromosome karyotype was found. In case 2, the fetal fluorescence in situ hybridization (FISH) results showed no abnormality. (2) During the first pregnancy of case 1, there was a 1.351 Mb of single-copy deletion in chromosome 17q12 (34 817 422-36 168 104) and a 1.187 Mb of single-copy duplication in chromosome 3p26.3 (838 934-2 026 269) extracted from umbilical cord blood. Moreover, a 1.299 Mb of single copy duplication in chromosome 3p26.3 (726 645-2 026 269) extracted from maternal peripheral blood was detected. (3) DNA analysis of the umbilical cord blood of case 2 showed a 1.351 Mb of single copy deletion in 17q12. No abnormal copy number variants (CNVs) were detected in the peripheral DNA of the couple. Conclusions Invasive prenatal detection of CNVs in cases with abnormal fetal kidney ultrasound findings might help to confirm the diagnosis and guide genetic counseling.

Background Phenylketonuria (PKU) is a genetic metabolic disease with a relatively higher incidence, but only a few studies about the prenatal diagnosis of PKU have been reported so far in China. The aim of this study was to characterize the spectrum of mutations in PAH gene in PKU probands and the prenatal diagnosis of PKU in north China.Methods A total of 157 families in which PKU patients had been diagnosed were included in the study. The 13 exons and their flanking sequences of PAH gene were amplified by PCR and sequenced in the probands. If none or only one mutant allele was found in the probands, the sample was subjected to MLPA for large deletions/duplication detection in PAH gene. Prenatal diagnosis was performed for pregnant women in these families.Results Pathogenic mutation in PAH was found in 2 alleles in 148 probands and in one allele in 7 probands, and the mutation was not detected in 2 probands. There were 289 point mutants, 10 frame-shift mutations and 4 large deletions with a total of 80 kinds of mutations. The most prevalent mutations were R243Q (17.2%), EX6-96A>G (8.6%) and V399V (8.0%). We also found a novel mutation of 163_164insATAT. Prenatal diagnosis of 95 families found 21 healthy fetus (20.4%), 52 carriers (50.5%) and 30 patients (29.1%), and the accuracy of prenatal diagnosis was confirmed after birth of the fetuses.Conclusion We present here a spectrum of mutations in PAH gene in PKU patients in north China. Prenatal diagnosis for PKU is useful for PKU families to prevent birth of another PKU case.

Purpose: 1p36 deletion syndrome is the most common terminal deletion syndrome, with an incidence of 1/5,000 newborns. But 22q13 duplications seem to be exceedingly rare. A combined 1p36 deletion and 22q13 duplication was more rarely observed and presented variability of clinical features, which increases the importance of reporting additional cases in order to better characterize genotype-phenotype correlations. Methods: A boy and a fetus from a single family with combined 1p36 deletion and 22q13 duplication characterized by array CGH and MLPA were described here. Results: The proband presented severe developmental delay, hypotonia, epilepsy, feeding difficulties with failure to thrive, tracheal malformation, clinodactyly, strephexopodia, fair skin, facial dysmorphism and died at 1 year of age. Array CGH uncovered a 9.3-Mb deletion of 1p36 plus a 6.655-Mb duplication of 22q13in the proband. This rearrangement was confirmed by MLPA. When his mother was pregnant again, array CGH detected an almost identical rearrangement with that of the proband. She terminated the pregnancy at 24 weeks gestation. The fetus was female and had similar facial dysmorphism as the proband. Karyotypes of parents are all normal. Conclusions: Most of the features in the proband were similar to those associated with both isolated 1p36 deletions and 22q13 duplications. However, tracheal malformation, fair skin and strephexopodia were only observed in our patient. Given that 22q13 duplications are rare and not as well characterized as 1p36 deletions, we attributed these features to 22q13 duplications. Importantly, we emphasize importance of prenatal diagnosis of females who had such abnormal pregnancy.

目的:探讨肾小球线粒体动力相关蛋白1(Drp1)、P-Drp1 (Ser616)和线粒体分裂蛋白1(Fis1)表达与足细胞损伤及蛋白尿发生的关系. 方法:建立阿霉素大鼠肾病模型,用免疫组化和western blot检测肾小球和肾皮质Drp1 、P-Drp1 (Ser616)及Fis1的表达,分析上述蛋白表达与蛋白尿及足细胞线粒体形态的相关性.在小鼠足细胞系MPC5过表达Drp1,分析对凋亡和线粒体形态的影响. 结果:肾小球和肾皮质Drp1在阿霉素大鼠肾病模型4周和6周时表达增强,肾小球P-Drp1(Ser616)在6周时表达增强,肾小球Fis1在2周和6周时增强.肾小球和肾皮质Drp1、肾小球P-Drp1 (Ser616)和Fis1表达与24h尿蛋白正相关.肾小球Drp1表达量与足细胞线粒体胞浆密度和线粒体细胞密度呈负相关.肾小球P-Drp1 (Ser616)与足细胞线粒体最大长宽比呈负相关.肾小球Fis1与足细胞线粒体面积和周长呈负相关.过表达Drp1致小鼠足细胞凋亡显著增多,线粒体片段化. 结论:肾小球Drp1、P-Drp1(Ser616)与Fis1高表达参与阿霉素大鼠肾病模型蛋白尿的发生,Drp1高表达致线粒体片段化和足细胞凋亡.

Objective: Preeclampsia is a pregnancy-related syndrome. Shallow invasion of uterine wall by trophoblast cells has been generally accepted as the major pathological change of this disorder. We previously found downregulation of miR-195 in preeclamptic placentas. Bioinformatic analysis predicted a type II activin receptor, activin receptor type-2B (ActRIIB), as one of the potential targets of miR-195. Considering the key function of activin A on trophoblast cell behaviors and placenta development, we proposed miR-195 may affect trophoblast cell invasion by repressing the expression of ActRIIB. Methods: The colocalization of ActRIIB and miR-195 in human placenta was measured by in-situ hybridization and immunohistochemistry. Western blotting, real-time PCR and dual luciferase assay were performed in human trophoblast cell line, HTR8/SVneo cells, to validate the targeting of ActRIIB by miR-195. Cell invasiveness was analyzed using transwell insert invasion assay in HTR8/SVneo cells. Results: In human placenta, ActRIIB and miR-195 exhibited similar localization in various subtypes of trophoblast cells, including villous and extravillous trophoblasts. The protein expressions of ActRIIB in preeclamptic placenta were significantly higher as compared with the normal controls, which was opposite to the changing pattern of miR-195. In HTR8/SVneo cells, miR-195 could directly target and suppress the expression of ActRIIB. Meanwhile, the invasion-promoting effect of miR-195 on trophoblast cells could be largely impeded by ActRIIB overexpression. Conclusion: In human trophoblast cells, miR-195 could promote cell invasion via directly targeting ActRIIB. The impaired miR-195 expression may contribute to the occurrence or development of preeclampsia through interfering with activin/nodal signaling in the placenta.

Background: Wolf-Hirschhorn syndrome (WHS) is a contiguous gene syndrome that is typically caused by a deletion of the distal portion of the short arm of chromosome 4. However, there are few reports about the features of Chinese WHS patients. This study aimed to characterize the clinical and molecular cytogenetic features of Chinese WHS patients using the combination of multiplex ligation-dependent probe amplification (MLPA) and array comparative genomic hybridization (array CGH).Methods: Clinical information was collected from ten patients with WHS. Genomic DNA was extracted from the peripheral blood of the patients. The deletions were analyzed by MLPA and array CGH.Results: All patients exhibited the core clinical symptoms of WHS, including severe growth delay, a Greek warrior helmet facial appearance, differing degrees of intellectual disability, and epilepsy or electroencephalogram anomalies. The 4p deletions ranged from 2.62 Mb to 17.25 Mb in size and included LETM1, WHSC1, and FGFR3.Conclusions: The combined use of MLPA and array CGH is an effective and specific means to diagnose WHS and allows for the precise identification of the breakpoints and sizes of deletions. The deletion of genes in the WHS candidate region is closely correlated with the core WHS phenotype.

Introduction: Chromosome Xq28 duplications encompassing methyl-CpG-binding protein 2 gene (MECP2) are observed most in males with a severe neurodevelopmental disorder associated with hypotonia, spasticity, severe learning disability, delayed psychomotor development, and recurrent pulmonary infections. Most female carriers are asymptomatic due to extremely or completely skewed X-inactivation.Methods: A retrospective clinical and molecular study was conducted to examine 16 patients and two fetuses from 10 families who were identified among patients with Xq28 duplications who presented at genetic clinics.Results: Of all 16 patients, 10 had a family history. Only one patient was female. All of the patients had no relevant pre-natal history. All of the patients exhibited severe psychomotor developmental delay, infantile hypotonia and recurrent infections. Some of the patients exhibited cardiac abnormalities, gastrointestinal mobility problems, hydrocele of tunica vaginalis, cryptorchidism, and autistic phenotypes. Additionally, neonatal kidney calculus, premature closure of the fontanel and pulmonary sequestration were found in the patients. Duplication sizes in these patients range from 0.21 to 14.391 Mb (most were smaller than 1 Mb), and all the duplications included host cell factor C1 (HCFC1), interleukin-1 receptor-associated kinase 1 (IRAK1), and MECP2. Bioinformatics analysis revealed that approximately half of the distal breakpoints were located within the low-copy repeats (LCRs), which may be involved in the recombination. The two fetuses were found to be healthy in the prenatal diagnosis.Conclusion: This is the first large cohort of patients with MECP2 duplication syndrome, including a female, reported in China. Interestingly, neonatal kidney calculus, premature closure of the fontanel and pulmonary sequestration were first reported in this syndrome. However, it was difficult to distinguish if these patients represented unique cases or if these phenotypes can be considered as part of the syndrome. The correlation between the infrequent phenotypes and duplications/genes in the duplication region needs further systematic delineation. In conclusion, our study suggested that it is important to emphasize molecular genetic analysis in patients with developmental delay/intellectual disability and recurrent infections and that it is especially important for familial female carriers to accept prenatal diagnosis. (C) 2016 Elsevier Masson SAS. All rights reserved.

Objective: We present prenatal diagnosis, genetic counseling, and molecular cytogenetic features of familial recurrence of Wolf-Hirschhorn syndrome (WHS).Materials and methods: A 31-year-old woman was referred to a hospital at 24 weeks of gestation because of abnormal ultrasound findings in the fetus. Her first child was a boy who had growth retardation, mental defect, and a distinctive facial appearance. Based on the conventional cytogenetic analysis, the combined use of multiplex ligation-dependent probe amplification (MLPA) and array comparative genomic hybridization (aCGH) facilitated the prenatal diagnosis and genetic counseling in the fetus. Results of the standard G-banging karyotype analysis of the fetus, the parents, and the boy were normal.Results: The MLPA analysis revealed the same 4p microdeletion accompanied by 2p microduplication in the fetus and the boy. The aCGH analysis revealed a 3.57-Mb 4p16.3 microdeletion or arr [hg19] 4p16.3 (71,552-3,636,893) x1 in the fetus and a 3.29-Mb 4p16.3 microdeletion or arr [hg19] 4p16.3 (71,148-3,360,737) xl in the boy. The 3.57-Mb 4p16.3 microdeletion encompassed 39 OMIM genes. The 3.29-Mb 4p16.3 microdeletion encompassed 36 OMIM genes. They both included LETM1 and WHSC1. The 2p25.3 microduplication was smaller than 666 kb and encompassed only one OMIM gene, ACP1.Conclusion: The combined use of MLPA and aCGH is an effective way to diagnose recurrent WHS. Although WHS is typically caused by a de novo deletion, prenatal diagnosis and genetic counseling are necessary in the next pregnancy in families that have suffered such cases. Copyright (C) 2016, Taiwan Association of Obstetrics & Gynecology. Published by Elsevier Taiwan LLC.

OBJECTIVE:To investigate whether the four boys with delayed motor development and intellectual disability suffer from MECP 2 duplication syndrome.METHOD:Blood specimens and clinical data of four patients and mothers of patient 2 and patient 4 were collected. Genomic DNA was extracted from peripheral blood using DNA extraction kit. At first multiplex ligation-dependent probe amplification (MLPA) was employed in 4 patients, two distinct kits SALSA P036 and P070 for sub-telomere screening, and SALSA P245 for the 22 common microdeletion and microduplication syndromes. Then array-CGH analysis was carried out. Two mothers of patients were tested by array- comparative genomic hybridization (CGH) and X chromosome inactivation analysis.RESULT:All the 4 patients presented with severe hypotonia, delayed motor development, intellectual disability and absent or limited language. Three patients manifested recurrent pneumonia in infancy except patient 2. Four patients had duplication on chromosome Xq28 with MLPA kit SALSA P245. Array-CGH identified the size of each duplication on Xq28. The precise size of each duplication was different in the four patients: patient 1, 14.931 Mb, patient 2, 0.393 Mb, patient 3, 0.482 Mb and patient 4, 0.299 Mb. To compare Xq28 duplications with UCSC database (http://genome.ucsc.edu/) revealed that each duplication harbors the MECP 2 and HCFC 1 gene. Mothers of patient 2 and patient 4 also carried microduplication on Xq28. X chromosome inactivation analysis demonstrated completely skewed inactivation (0: 100) and it is the inactive allele that passed on to the patients.CONCLUSION:For patients that present with delayed motor development, intellectual disability, hypotonia, absent or limited language and recurrent infection, combination of MLPA and array- CGH is effective and specific diagnostic methods of MECP 2 duplication syndrome.

The genotype-phenotype relationship in diseases with mtDNA point mutations is still elusive. The maintenance of wild-type mtDNA copy number is essential to the normal mitochondrial oxidative function. This study examined the relationship between mtDNA copy number in blood and urine and disease severity of the patients harboring A3243G mutation. We recruited 115 A3243G patients, in which 28 were asymptomatic, 42 were oligo-symptomatic, and 45 were poly-symptomatic. Increase of total mtDNA copy number without correlation to the proportion of mutant mtDNA was found in the A3243G patients. Correlation analyses revealed that wild-type mtDNA copy number in urine was the most important factor correlated to disease severity, followed by proportion of mutant mtDNA in urine and proportion of mutant mtDNA in blood. Wild-type copy number in urine negatively correlated to the frequencies of several major symptoms including seizures, myopathy, learning disability, headache and stroke, but positively correlated to the frequencies of hearing loss and diabetes. Besides proportion of mutant mtDNA in urine, wild-type copy number in urine is also an important marker for disease severity of A3243G patients.