Desheng Liang

A SNP in the ABCC11 gene is the determinant of human earwax type

Human earwax consists of wet and dry types. Dry earwax is frequent in East Asians, whereas wet earwax is common in other populations. Here we show that a SNP, 538G → A (rs17822931), in the ABCC11 gene is responsible for determination of earwax type. The AA genotype corresponds to dry earwax, and GA and GG to wet type. A 27-bp deletion in ABCC11 exon 29 was also found in a few individuals of Asian ancestry. A functional assay demonstrated that cells with allele A show a lower excretory activity for cGMP than those with allele G. The allele A frequency shows a north-south and east-west downward geographical gradient; worldwide, it is highest in Chinese and Koreans, and a common dry-type haplotype is retained among various ethnic populations. These suggest that the allele A arose in northeast Asia and thereafter spread through the world. The 538G → A SNP is the first example of DNA polymorphism determining a visible genetic trait.

Non‐invasive prenatal testing of fetal whole chromosome aneuploidy by massively parallel sequencing

To determine whether non‐invasive prenatal testing by maternal plasma DNA sequencing can uncover all fetal chromosome aneuploidies in one simple sequencing event.

Molecular analysis of hearing loss associated with enlarged vestibular aqueduct in the mainland Chinese: a unique SLC26A4 mutation spectrum.

AbstractIt has been shown that mutations in the SLC26A4 gene are involved in syndromic deafness characterized by congenital sensorineural hearing impairment and goitre (Pendreds syndrome), as well as in congenital isolated deafness (DFNB4), both of which are associated with enlarged vestibular aqueduct (EVA). The prevalence of SLC26A4 mutations in Pendreds syndrome is clearly established in many ethnic groups, but the data from Mainland Chinese patients with deafness and EVA remain poor. In this report, 15 patients from 13 unrelated Chinese families with deafness and EVA were analyzed for SLC26A4 using direct sequencing. A total of 15 pathogenic mutations were observed in 11 unrelated families, 4 of which were novel. One mutation, IVS7-2A>G, was most common, accounting for 22.3% (5/22) of all the mutant alleles, and H723R was infrequent. To date, a total of 23 mutations have been reported among the Chinese, 13 of which were unique. In conclusion, EVA could be a radiological marker for SLC26A4 analysis among Mainland Chinese hearing-loss patients, and the SLC26A4 mutation spectrum in the Chinese was different from other reported populations.

Mutation in PITX2 is associated with ring dermoid of the cornea

Ring dermoid of the cornea (RDC, MIM180550) is an autosomal dominantly inherited syndrome characterised by bilateral annular limbal dermoids with corneal and conjunctival extension. The genetic basis of RDC is unknown. We report linkage of chromosome 4q24-q26 to RDC and identification of a missense mutation in PITX2 in 17 disease affected individuals but not in eight genetically related normal individuals in a large Chinese family. A large Chinese family with 17 individuals affected by the RDC was identified (figs 1 and 2). All patients were diagnosed by the same physician (XHX). Informed written consent for blood sample collection was obtained from all participants. Figure 1  Eyes affected by ring dermoid of the cornea (RDC) in two patients, II-2 (upper panel) and IV-3 (lower panel). Yellow-white tumour-like apophyses are visible on the corneal border of both eyes. The apophyses are diffuse in the superficial layer of the cornea and conjunctiva. The corneal border is not clear and the diameter of the transparent region of cornea is diminished to about 7–8 mm. The upward shift of the right pupil of IV-3 is caused by cataract resection (the affected individual, IV-3, also has bilateral glaucoma and congenital cataracts in the right eye). Figure 2  Recombination analysis of the family with ring dermoid of the cornea (RDC). Pedigree of the family affected by RDC and haplotype analysis for 14 markers on 4q22-q26. Markers (from top to bottom) are centromere-D4S1560-D4S2966-D4S1572-D4S1570-D4S1564-D4S2945-D4S2989-D4S1616-D4S406-D4S193-D4S1613-D4S1522-D4S1612-D4S427-telomere. The haplotype co-segregating with the disorder is boxed. Linkage analysis and genotyping were done essentially as previously described.1 Genome-wide screening was carried out with 382 microsatellite markers covering all autosomal chromosomes, with an average interval of 10 cM (ABI PRISM™ linkage mapping set, version 2.0). Fine …

TALE nickase mediates high efficient targeted transgene integration at the human multi-copy ribosomal DNA locus.

Although targeted gene addition could be stimulated strikingly by a DNA double strand break (DSB) created by either zinc finger nucleases (ZFNs) or TALE nucleases (TALENs), the DSBs are really mutagenic and toxic to human cells. As a compromised solution, DNA single-strand break (SSB) or nick has been reported to mediate high efficient gene addition but with marked reduction of random mutagenesis. We previously demonstrated effective targeted gene addition at the human multicopy ribosomal DNA (rDNA) locus, a genomic safe harbor for the transgene with therapeutic potential. To improve the transgene integration efficiency by using TALENs while lowering the cytotoxicity of DSBs, we created both TALENs and TALE nickases (TALENickases) targeting this multicopy locus. A targeting vector which could integrate a GFP cassette at the rDNA locus was constructed and co-transfected with TALENs or TALENickases. Although the fraction of GFP positive cells using TALENs was greater than that using TALENickases during the first few days after transfection, it reduced to a level less than that using TALENickases after continuous culture. Our findings showed that the TALENickases were more effective than their TALEN counterparts at the multi-copy rDNA locus, though earlier studies using ZFNs and ZFNickases targeting the single-copy loci showed the reverse. Besides, TALENickases mediated the targeted integration of a 5.4 kb fragment at a frequency of up to 0.62% in HT1080 cells after drug selection, suggesting their potential application in targeted gene modification not being limited at the rDNA locus.

Noninvasive Prenatal Testing for Wilson Disease by Use of Circulating Single-Molecule Amplification and Resequencing Technology (cSMART)

BACKGROUND Noninvasive prenatal testing (NIPT) for monogenic diseases by use of PCR-based strategies requires precise quantification of mutant fetal alleles circulating in the maternal plasma. The study describes the development and validation of a novel assay termed circulating single-molecule amplification and resequencing technology (cSMART) for counting single allelic molecules in plasma. Here we demonstrate the suitability of cSMART for NIPT, with Wilson Disease (WD) as proof of concept. METHODS We used Sanger and whole-exome sequencing to identify familial ATP7B (ATPase, Cu(++) transporting, β polypeptide) gene mutations. For cSMART, single molecules were tagged with unique barcodes and circularized, and alleles were targeted and replicated by inverse PCR. The unique single allelic molecules were identified by sequencing and counted, and the percentage of mutant alleles in the original maternal plasma sample was used to determine fetal genotypes. RESULTS Four families with WD pedigrees consented to the study. Using Sanger and whole-exome sequencing, we mapped the pathogenic ATP7B mutations in each pedigree and confirmed the probands original diagnosis of WD. After validation of cSMART with defined plasma models mimicking fetal inheritance of paternal, maternal, or both parental mutant alleles, we retrospectively showed in second pregnancies that the fetal genotypes assigned by invasive testing and NIPT were concordant. CONCLUSIONS We developed a reliable and accurate NIPT assay that correctly diagnosed the fetal genotypes in 4 pregnancies at risk for WD. This novel technology has potential as a universal strategy for NIPT of other monogenic disorders, since it requires only knowledge of the parental pathogenic mutations.

Copy Number Variation Sequencing for Comprehensive Diagnosis of Chromosome Disease Syndromes

Detection of chromosome copy number variation (CNV) plays an important role in the diagnosis of patients with unexplained clinical symptoms and for the identification of chromosome disease syndromes in the established fetus. In current clinical practice, karyotyping, in conjunction with array-based methods, is the gold standard for detection of CNV. To increase accessibility and reduce patient costs for diagnostic CNV tests, we speculated that next-generation sequencing methods could provide a similar degree of sensitivity and specificity as commercial arrays. CNV in patient samples was assessed on a medium-density single nucleotide polymorphism array and by low-coverage massively parallel CNV sequencing (CNV-seq), with mate pair sequencing used to confirm selected CNV deletion breakpoints. A total of 10 ng of input DNA was sufficient for accurate CNV-seq diagnosis, although 50 ng was optimal. Validation studies of samples with small CNVs showed that CNV-seq was specific and reproducible, suggesting that CNV-seq may have a potential genome resolution of approximately 0.1 Mb. In a blinded study of 72 samples with known gross and submicroscopic CNVs originally detected by single nucleotide polymorphism array, there was high diagnostic concordance with CNV-seq. We conclude that CNV-seq is a viable alternative to arrays for the diagnosis of chromosome disease syndromes.

Pregnancy-related complications and adverse pregnancy outcomes in multiple pregnancies resulting from assisted reproductive technology: a meta-analysis of cohort studies

OBJECTIVE To provide an up-to-date comparison of pregnancy-related complications and adverse pregnancy outcomes of multiple pregnancies generated with assisted reproductive technology (ART) vs. spontaneous conception. DESIGN Meta-analysis. SETTING University-affiliated teaching hospital. PATIENT(S) Multiple pregnancies conceived by ART or naturally. INTERVENTION(S) Searches through October 2014 were conducted on PubMed, Google Scholar, Cochrane Libraries, China Biology Medicine disc, Chinese Scientific Journals Fulltext Database, China National Knowledge Infrastructure, and Wanfang Data, to identify studies that met prestated inclusion criteria. Either a fixed- or a random-effects model was used to calculate the overall combined risk estimates. Subgroup analysis was performed to explore potential heterogeneity moderators. MAIN OUTCOME MEASURE(S) Pregnancy-related complications and adverse pregnancy outcomes. RESULT(S) Thirty-nine cohort studies involving 146,008 multiple births were included in the meta-analysis. Multiple pregnancies from ART were associated with a higher risk of premature rupture of membranes (relative risk [RR] = 1.20, 95% confidence interval [CI]: 1.05-1.37; I(2) = 15%); pregnancy-induced hypertension (RR = 1.11, 95% CI: 1.04-1.19; I(2) = 6%); gestational diabetes mellitus (RR = 1.78, 95% CI: 1.25-2.55; I(2) = 42%); preterm birth (RR = 1.08, 95% CI: 1.03-1.14; I(2) = 83%); very preterm birth (RR = 1.18, 95% CI: 1.04-1.34; I(2) = 79%); low birth weight (RR = 1.04, 95% CI: 1.01-1.07; I(2) = 47%); very low birth weight (RR = 1.13, 95% CI: 1.01-1.25; I(2) = 62%); and congenital malformation (RR = 1.11, 95% CI: 1.02-1.22; I(2) = 30%). The relevant heterogeneity moderators have been identified by subgroup analysis. Sensitivity analysis yielded similar results. No evidence of publication bias was observed. CONCLUSION(S) Although the role of potential bias and evidence of heterogeneity should be carefully evaluated, the present study suggests that multiple pregnancies generated via ART, vs. spontaneous conception, are associated with higher risks of pregnancy-related complications and adverse pregnancy outcomes. Further research is needed to determine which aspect of ART poses the most risk and how this risk can be minimized.

A ZRS duplication causes syndactyly type IV with tibial hypoplasia

A ZRS Duplication Causes Syndactyly Type IV With Tibial Hypoplasia Lingqian Wu,* Desheng Liang, Norio Niikawa, Fen Ma, Miao Sun, Qian Pan, Zhigao Long, Zhongmin Zhou, Koh-ichiro Yoshiura, Hua Wang, Daisuke Sato, Gen Nishimura, Heping Dai, Xue Zhang, and Jiahui Xia National Laboratory of Medical Genetics, Central South University, Changsha, Hunan, China Solution Oriented Research of Science and Technology (SORST), Japan Science and Technology Agency (JST), Kawaguchi, Japan Research Institute of Personalized Health Sciences, Health Sciences University of Hokkaido, Hokkaido, Japan McKusick-Zhang Center for Genetic Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China Department of Human Genetics, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan Women and Children’s Hospital of Hunan Province, Changsha, Hunan, China Department of Radiology, Tokyo Metropolitan Kiyose Children’s Hospital, Tokyo, Japan

A syndactyly type IV locus maps to 7q36

AbstractSyndactyly occurs as an isolated abnormality or a part of a malformation syndrome. Syndactyly types I, II, III and V have been mapped to chromosomal regions 2q34-q36, 2q31-q32, 6q21-q23.2 and 2q31-q32, respectively, whereas syndactyly type IV (SD4) is extremely rare, and its gene localization has not yet been assigned. The SD4 manifests complete syndactyly of all fingers accompanied with polydactyly, and flexion of the fingers gives the hand a cup-shaped appearance. We performed a linkage and haplotype analysis of a Chinese pedigree with autosomal dominant, non-syndromic SD4 using a set of 406 microsatellite markers. The analysis gave the maximum two-point LOD score of 1.613 at recombination fraction of 0.00 and penetrance of 1.00. Thus, the SD4 locus in the family was likely assigned to a 17.39-cM region at a segment between markers D7S3070 and D7S559 at 7q36, although the LOD score obtained was not high enough to conclude the localization. Analysis of three candidate genes, LMBR1, SHH and ZRS, failed to identify any pathogenic mutations. Our gene mapping may give a clue to identify the putative SD4 gene and provide a better understanding of normal human limb development.