Inherited and multiple de novo mutations in autism/developmental delay risk genes suggest a multifactorial model.

Background: We previously performed targeted sequencing of autism risk genes in probands from the Autism Clinical and Genetic Resources in China (ACGC) (phase I). Here, we expand this analysis to a larger cohort of patients (ACGC phase II) to better understand the prevalence, inheritance, and genotype-phenotype correlations of likely gene-disrupting (LGD) mutations for autism candidate genes originally identified in cohorts of European descent. Methods: We sequenced 187 autism candidate genes in an additional 784 probands and 85 genes in 599 probands using single-molecule molecular inversion probes. We tested the inheritance of potentially pathogenic mutations, performed a meta-analysis of phase I and phase II data and combined our results with existing exome sequence data to investigate the phenotypes of carrier parents and patients with multiple hits in different autism risk genes. Results: We validated recurrent, LGD, de novo mutations (DNMs) in 13 genes. We identified a potential novel risk gene (ZNF292), one novel gene with recurrent LGD DNMs (RALGAPB), as well as genes associated with macrocephaly (GIGYF2 and WDFY3). We identified the transmission of private LGD mutations in genes predominantly associated with DNMs and showed that parental carriers tended to share milder autism-related phenotypes. Patients that carried DNMs in two or more candidate genes show more severe phenotypes. Conclusions: We identify new risk genes and transmission of deleterious mutations in genes primarily associated with DNMs. The fact that parental carriers show milder phenotypes and patients with multiple hits are more severe supports a multifactorial model of risk.

A novel NHS mutation causes Nance-Horan Syndrome in a Chinese family.

BACKGROUND: Nance-Horan Syndrome (NHS) (OMIM: 302350) is a rare X-linked developmental disorder characterized by bilateral congenital cataracts, with occasional dental anomalies, characteristic dysmorphic features, brachymetacarpia and mental retardation. Carrier females exhibit similar manifestations that are less severe than in affected males. METHODS: Here, we report a four-generation Chinese family with multiple affected individuals presenting Nance-Horan Syndrome. Whole-exome sequencing combined with RT-PCR and Sanger sequencing was used to search for a genetic cause underlying the disease phenotype. RESULTS: Whole-exome sequencing identified in all affected individuals of the family a novel donor splicing site mutation (NM_198270: c.1045 + 2T > A) in intron 4 of the gene NHS, which maps to chromosome Xp22.13. The identified mutation results in an RNA processing defect causing a 416-nucleotide addition to exon 4 of the mRNA transcript, likely producing a truncated NHS protein. CONCLUSIONS: The donor splicing site mutation NM_198270: c.1045 + 2T > A of the NHS gene is the causative mutation in this Nance-Horan Syndrome family. This research broadens the spectrum of NHS gene mutations, contributing to our understanding of the molecular genetics of NHS.

Phenotypic expansion of the interstitial 16p13.3 duplication: a case report and review of the literature.

Genotype-phenotype analysis of at least 25 individuals with interstitial 16p13.3 duplications defines a recognizable syndrome associated with duplication of a critical Rubinstein-Taybi region encompassing only the CREBBP gene. Nevertheless, variable or incompletely penetrant phenotype has been reported previously. We here report a case of a 5-year old boy with a recognizable phenotype of this syndrome, including intellectual disability, mild arthrogryposis, small and proximally implanted thumbs and characteristic facial features. In addition, growth delay, microcephaly and distinguishable structural brain MRI abnormalities were observed. A de novo 1.5 Mb interstitial duplication of 16p13.3 was detected by SNP-array and fluorescence in situ hybridization (FISH). Short tandem repeat polymorphism (STRP) analysis with marker D16S475 indicated that the duplication was formed before maternal meiosis II. Our findings highlight the variable clinical features and further expand the phenotypic spectrum correlated with this lately proposed syndrome.

Disruption of Contactin 4 in two subjects with autism in Chinese population.

Autism is a heterogeneous childhood neurodevelopmental disorder that is characterised by deficits in verbal communication, impaired social interactions, restricted interests and repetitive behaviours. Using an Illumina HumanCNV370-Quad BeadChip, we identified two Han Chinese individuals with autism and large duplications (~1.6 Mb and ~2.4 Mb) disrupting the same CNTN4 gene. CNTN4 encodes a protein that functions as a cell-adhesion molecule and may play an essential role in the formation of axon connections in the developing nervous system. The disruption of this gene has been reported to be the cause of the 3p deletion syndrome and also a possible susceptibility factor for autism spectrum disorders (ASDs). Our results suggest that rare copy number variations (CNVs) in CNTN4 may also influence autism susceptibility in Asian populations. Interestingly, a comparison of the clinical phenotypes between the two subjects revealed that the subject with the 2.4 Mb CNV (involving several other genes) presented with a more severe phenotype than the subject with the 1.6 Mb CNV (disrupting only CNTN4 and CNTN6). This suggests that other genes in the nearby region may contribute to the pathogenesis.

A novel splicing mutation in COL1A1 gene caused type I osteogenesis imperfecta in a Chinese family.

Osteogenesis imperfect (OI) is a heritable connective tissue disorder with bone fragility as a cardinal manifestation, accompanied by short stature, dentinogenesis imperfecta, hyperlaxity of ligaments and skin, blue sclerae and hearing loss. Dominant form of OI is caused by mutations in the type I procollagen genes, COL1A1/A2. Here we identified a novel splicing mutation c.3207+1G>A (GenBank ID: JQ236861) in the COL1A1 gene that caused type I OI in a Chinese family. RNA splicing analysis proved that this mutation created a new splicing site at c.3200, and then led to frameshift. This result further enriched the mutation spectrum of type I procollagen genes.

[Linkage analysis of susceptibility loci in 2 target chromosomes in pedigrees with paranoid schizophrenia and undifferentiated schizophrenia].

OBJECTIVE: To investigate the relationship of susceptibility loci in chromosomes 1q21-25 and 6p21-25 and schizophrenia subtypes in Chinese population. METHODS: A genomic scan and parametric and non-parametric analyses were performed on 242 individuals from 36 schizophrenia pedigrees, including 19 paranoid schizophrenia and 17 undifferentiated schizophrenia pedigrees, from Henan province of China using 5 microsatellite markers in the chromosome region 1q21-25 and 8 microsatellite markers in the chromosome region 6p21-25, which were the candidates of previous studies. All affected subjects were diagnosed and typed according to the criteria of the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revised (DSM-IV-TR; American Psychiatric Association, 2000). All subjects signed informed consent. RESULTS: In chromosome 1, parametric analysis under the dominant inheritance mode of all 36 pedigrees showed that the maximum multi-point heterogeneity Log of odds score method (HLOD) score was 1.33 (α = 0.38). The non-parametric analysis and the single point and multi-point nonparametric linkage (NPL) scores suggested linkage at D1S484, D1S2878, and D1S196. In the 19 paranoid schizophrenias pedigrees, linkage was not observed for any of the 5 markers. In the 17 undifferentiated schizophrenia pedigrees, the multi-point NPL score was 1.60 (P= 0.0367) at D1S484. The single point NPL score was 1.95(P= 0.0145) and the multi-point NPL score was 2.39 (P= 0.0041) at D1S2878. Additionally, the multi-point NPL score was 1.74 (P= 0.0255) at D1S196. These same three loci showed suggestive linkage during the integrative analysis of all 36 pedigrees. In chromosome 6, parametric linkage analysis under the dominant and recessive inheritance and the non-parametric linkage analysis of all 36 pedigrees and the 17 undifferentiated schizophrenia pedigrees, linkage was not observed for any of the 8 markers. In the 19 paranoid schizophrenias pedigrees, parametric analysis showed that under recessive inheritance mode the maximum single-point HLOD score was 1.26 (α = 0.40) and the multi-point HLOD was 1.12 (α = 0.38) at D6S289 in the chromosome 6p23. In nonparametric analysis, the single-point NPL score was 1.52 (P= 0.0402) and the multi-point NPL score was 1.92 (P= 0.0206) at D6S289. CONCLUSION: Susceptibility genes correlated with undifferentiated schizophrenia pedigrees from D1S484, D1S2878, D1S196 loci, and those correlated with paranoid schizophrenia pedigrees from D6S289 locus are likely present in chromosome regions 1q23.3 and 1q24.2, and chromosome region 6p23, respectively.

FBXO7 gene mutations may be rare in Chinese early-onset Parkinsonism patients.

A recent study has shown that FBXO7 is a causative gene for PARK15-linked autosomal recessive early-onset Parkinsonism which was described by Davison for the first time in 1954 and known as Pallido-Pyramidal Disease or Parkinsonia-Pyramidal Syndrome in the past. In order to investigate the characteristics of FBXO7 gene mutations in Chinese early-onset Parkinsonism patients, we performed polymerase chain reaction and DNA direct sequencing on 135 patients and 200 controls. In this study, we found 10 polymorphisms including two novel polymorphisms (-274G-->C, c.A155G), but no pathogenetic mutations in the FBXO7 gene were detected. This suggests that FBXO7 mutations may be rare in Chinese early-onset Parkinsonism patients.

Association analysis of CNTNAP2 polymorphisms with autism in the Chinese Han population.

OBJECTIVES: Autism is a neurodevelopmental disorder, and genetic factors play an important role in its pathogenesis. Earlier findings suggest the CNTNAP2 as a predisposition locus of autism, but no study has been carried out on the possible association of CNTNAP2 with autism in the Chinese Han population. METHODS: In this study, three single nucleotide polymorphisms located within the CNTNAP2 were genotyped in 185 Chinese Han autistic families by polymerase chain reaction-restriction fragment length polymorphism analysis, followed by a transmission disequilibrium test. RESULTS: The results show that a common noncoding variant (rs10500171) is associated with the increased risk for autism, and haplotype T-A (rs7794745- rs10500171, P=0.011) and haplotype A-T-A (rs10244837- rs7794745- rs10500171, P=0.032) also showed evidence of association. CONCLUSION: The results of family-based association study suggested that the CNTNAP2 is a susceptibility gene of autism in the Chinese Han population.

[Identification of the small supernumerary marker chromosomes in two patients with Turner syndrome].

OBJECTIVE: To identify the small supernumerary marker chromosomes (sSMC) and guide the genetic counseling and medical treatment in two patients with Turner syndrome. METHODS: High resolution GTG and C banding, SRY amplification by PCR and fluorescence in situ hybridization (FISH) on metaphase chromosomes were performed to the two patients. RESULTS: The karyotypes of the two patients were 45, X [29]/46,X, +mar[31] and 45,X[71]/46,X, +mar[29] respectively. SRY test indicated SRY-positive for patient 1, whose sSMC was originated from chromosome Y. The karyotype was confirmed as 45,X[29]/46,X,idic(Y)(q10)[31]. ish idic(Y)(q10)(RP11-115H13x2) (SRY+) by FISH. While in patient 2, the sSMC was originated from chromosome X, whose karyotype was determined as 45, X[71]/46,X, r(X)(p11.23q21)[29]. ish r(X) (p11.23q21)(AL591394.11xAC092268.3). CONCLUSION: Using cytogenetic and molecular cytogenetic analyses, we have identified the sSMCs in two patients with Turner syndrome, which was helpful to the clinical diagnosis and treatment.

Analysis of SCA2 and SCA3/MJD repeats in Parkinson's disease in mainland China: genetic, clinical, and positron emission tomography findings.

To investigate the prevalence and clinical feature(s) of Parkinson's disease (PD) patients with expanded (ATXN2 and MJD1) genes of spinocerebellar ataxia type 2 and 3 (SCA2 and SCA3/MJD) in a mainland Chinese population, CAG triplet repeat expansions of (SCA2 and SCA3/MJD) genes (ATXN2 and MJD1) were analyzed in a cohort of 452 PD patients, including 386 sporadic and 66 familial forms. Striatal dopamine transporter was evaluated in two SCA2 and two SCA3/MJD-positive family members, an idiopathic PD patient and a healthy control using carbon (C11) [(11)C]-radiolabeled-CFT positron emission tomography (PET). We found two patients in one familial PD (FPD) family (1.5%) and two sporadic PD patients (0.5%) with expanded CAG repeats in the ATXN2 locus, four patients in two FPD families (3%) and another three sporadic PD patients (0.8%) in the MJD1 locus. [(11)C]-CFT PET in detected members in SCA2 and SCA3/MJD families showed decrements of (11)C-CFT uptake. These findings suggest that a mutation in SCA2 or SCA3/MJD may be one of the genetic causes of PD.

[Growing of human embryonic stem cells on feeders derived from themselves].

This study was carried out to determine whether mesenchymal stem cells (MSCs) derived from teratoma of human embryonic stem cells (hESCs) function as feeder cells to support hESCs growth. Approximately 5x10(6) hESCs were injected into the hind limb muscle of each SCID-beige mouse to form teratoma. After 8 weeks, the MSCs were isolated from the teratoma and cultured in Mesencult medium. Purified MSCs were then used as the feeder cells for hESCs culture. High purity MSCs derived from teratoma were isolated. The cells were morphologically similar to bone marrow MSCs (bMSCs). The teratoma-derived MSCs were negative for CD34 and CD45 but positive for CD29, CD49b, CD105, CD73, and CD90, which resembled those expressed by bMSCs. After passaged on MSCs feeder cells more than 10 passages, hESCs maintained hESC characteristics in morphology. Reverse PCR showed the expression of Oct4 and Nanog. SSEA-1 was negative and SSEA-4, TRA-1-60, and TRA-1-81 were positive. Alkaline phosphatase staining showed positive results.The karyotype remained normal. Moreover, the hECSs cultured on teratoma-derived MSCs formed teratoma in vivo and embryoid body in vitro confirmed their pluripotency. Accordingly, MSCs derived from hESCs by in vivo differentiation can be used as the feeder cells for hESCs culture.

[Mutation screening of the dystrophin gene in 14 Chinese Duchenne/Becker muscular dystrophy patients without gross deletions].

OBJECTIVE: To search for the dystrophin gene mutations of Duchenne muscular dystrophy (DMD) patients without gross deletions, in order to offer accurate genetic counseling and prenatal diagnosis for those families. METHODS: All 79 exons of the dystrophin gene as well as its 5'-UTR and 3'-UTR of 14 Chinese DMD/Becker muscular dystrphy (BMD) patients without detectable gross deletions were screened by denaturing high performance liquid chromatography (DHPLC) and heteroduplex fragments were identified by subsequent sequencing. RESULTS: Seven causative point mutations, including two novel ones, were detected in 7 patients. Fourteen known polymorphisms and 7 unknown intronic variations were also detected. Five mothers of the patients were obligate carriers. CONCLUSION: DHPLC is an efficient way of identifying point mutations and the female carriers in DMD families.

Screening of LRRK2 interactants by yeast 2-hybrid analysis.

OBJECTIVE: To isolate and identify the potential binding partners of LRRK2, a gene linked to both dominant familial form and sporadic form of Parkinson's disease, thus to further our knowledge of its function. METHODS: We used a sequence containing full-length of COR domain and part of ROC and MAPKKK domain as bait. The bait amplified by polymerase chain reaction (PCR) was then cloned into a yeast expression plasmid pGBKT7. After being sequenced and analyzed, pGBKT7-bait was transformed into the yeast strain AH109. Western blot was performed to confirm the expression of pGBKT7-bait in AH109 yeast strain. Then human fetal brain cDNA library was transformed into that yeast strain, which could express pGBKT7-bait fusion protein. The yeast strain which contained pGBKT7-bait and human fetal brain cDNA library was plated on quadruple dropout medium (SD/-Trp/-Leu/-His/-Ade) containing X-alpha-gal. We retested these positive colonies using 2 independent yeast strains AH109 contained pGBKT7-bait or pGBKT7, respectively. At last, these plasmids isolated from these true positive colonies were analyzed by bioinformatics. RESULTS: We obtained 9 true positive colonies, these colonies were sequenced, and we performed sequence Blast in GenBank. Three colonies of the 9 positive colonies were not in open reading-frames. Among other 6 colonies, there were known proteins including spermatid perinuclear RNA-binding protein (STRBP) and BCL2-associated athanogene 5 isoform b (BAG5), as well as unknown proteins including tyrosine phosphatase non-receptor type (PTPN23), l(3)mbt-like 3 isoform b (L3MBTL3), RALY RNA binding protein-like isoform 1 (RALYL), and Homo sapiens mRNA for KIAA1783 protein, partial cds (KIAA1783). CONCLUSION: True positive colonies of LRRK2 are successfully obtained by the yeast 2-hybrid. Our screened proteins may provide a new research clue for revealing biological functions of LRRK2, pathogenesis of Parkinson's disease, and other neurodegerations.

[Rapid prenatal diagnosis of chromosome aneuploidies in 60 uncultured amniotic fluid samples by fluorescence in situ hybridization].

OBJECTIVE: To evaluate the feasibility of rapid prenatal diagnosis of chromosome aneuploidies by interphase fluorescence in situ hybridization (FISH) using uncultured amniotic fluid. METHODS: Bacterial artificial chromosome (BAC) DNA probes were prepared and validated by using cultured peripheral blood. Interphase FISH for chromosomes 13, 18, 21, X and Y was performed in 60 amniotic fluid samples for the rapid prenatal diagnosis of chromosome aneuploidies, and the results were compared with the karyotypes from conventional cytogenetic analysis. RESULTS: Of all 60 cases, 58 were concordant with their karyotypes, and 1 case of inv(9) and another case of t(2,12) were identified by karyotyping. Two cases of trisomy 21 and 1 case of trisomy 18 were detected by FISH and confirmed with conventional cytogenetics (sensitivity=100%). There were no false-positive or false-negative results. CONCLUSION: This evaluation demonstrated that FISH employing BAC DNA probes could accurately and rapidly detect aneuploidies involving the above 5 chromosomes. However, as it does not identify structural chromosome aberrations and aneuploidies involving other chromosomes, it is not a substitute for conventional chromosome analysis, and the negative FISH result should be carefully interpreted.

[Biological characteristics and safety evaluation of endothelial progenitor cells from the umbilical cord blood].

OBJECTIVE: To investigate the biological characteristics of endothelial progenitor cells (EPCs) from the umbilical cord blood (UCB), and to evaluate their oncogenicity after long-term culture in vitro. METHODS: The mononuclear cells (MNCs) were isolated from the UCB and cultured in MCDB131 medium supplemented with 20% FBS, VEGF and other growth factors. Morphology of the EPCs was observed, and the growth curve of the EPCs was investigated. Surface antigens of the EPCs were analyzed by the flow-cytometer. The capability of intaking the acetylated low-density lipoprotein (acLDL) of the EPCs was detected using fluoresencent chemical method. The vasoformative capability and genetic stability of EPCs were cultured in matrigel, and examined by karyotype analysis. The oncogenicity of EPCs was verified by the tumorigenesis test in athymic mouse and soft agar. RESULTS: EPCs were successfully derived from the UCB, and could be passaged to at least 42(nd) generation and had strong abilities of proliferation, acLDL intake and vasoformation, but there was not oncogenicity. They expressed endothelial cell-surface antigens and maintained normal karyotype. CONCLUSION: The EPCs with proliferative potential can be isolated from the UCB. They can be passaged in long-term cultures without oncogenicity, and can maintain normal karyotype. The EPCs can be served as a new type of cells in cell and gene therapy.

[Azoospermia factor microdeletion on Y chromosome in patients with idiopathic azoospermia or severe oligozoospermia].

OBJECTIVE: To investigate the correlation between male infertility and Y chromosome microdeletions of azoospermia factor (AZF) regions, and to establish a reliable genetic diagnosis in idiopathic infertile male patients with azoospermia or severe oligozoospermia. METHODS: Multiplex PCR amplification of 6 sequence-tagged sites in AZF regions of the Y chromosome was examined among 100 normal karyotype male patients with azoospermia or oligozoospermia. RESULTS: Four patients (4%) had Y chromosome microdeletions, the microdeletions of 3 patients were idiopathic azoospermic and those of the other 1 patient were secretory azoospermia. CONCLUSION: The PCR-based Y chromosome microdeletion screening is simple and effective in the diagnosis of patients with severe male infertility. Microdeletion of Y chromosome is one of the major causes of severe dyszooospermia.