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ICGCG 2006 abstracts
The following are selected abstracts presented at the International Congress of Global Chinese Geneticists 2006 (ICGCG 2006). The congress, held in Beijing, August 2-4, 2006, was co-orgnized by Peking University, the Chinese Society of Medical Genetics, the Taiwan Human Genetics Society, the Hong Kong Society of Medical Genetics, the National Genome Institute of Singapore, Fudan University, Zhejiang University, and Wenzhou Medical College.
X-chromosome inactivation patterns in patients with Rett syndrome and the parental origin of priority inactivated X chromosome
Xin-Hua Bao, Shing-Ling Jiang, Fu-Ying Song, Hong Pan, Mei-Rong Li and Xi-Ru Wu
Department of Pediatrics, Peking University First Hospital, Beijing 100034, China
Background: Rett syndrome (RTT) is an X-link dominant neurodevelopment disorder. In order to well understand the relationship between the X chromosome inactivation, genotype and phenotype, X chromosome inactivation (XCI) patterns in patients with RTT and their mothers, parental origin of skewed X chromosome in RTT and the correlations of XCI with genotype/phenotype were analyzed.
Methods: XCI of 55 cases of RTT (52 cases with MECP2 mutations, no mutation was found in the other 3 cases), 53 RTT mothers and 48 normal females were analyzed with androgen receptor gene by methylation-sensitive restriction endonuclease HpaII, PCR and genescan.
Results: Eighty-two percent, 77% and 83% hetero-zygotes of the AR gene were detected in RTT patients, their mothers and the control group respectively. The X chromosome inactivation distribution pattern of RTT patients was significantly different from that of mothers and the control group (P<0.01). There was no difference between mothers and the control group in XCI distribution pattern (P>0.05). The rates of XCI in the area of 50:50 to 59:41 were higher in the mothers and the control group than in the RTT patients; the differences between them were statistically significant (P=0.019, 0.008). The non-random XCI in areas of ≥65:35 and ≥80:20 were 53.35% and 17.8% respectively in the RTT patients, compared with the mother group (36.6%, 7.3%) and the control group (35%, 10%). It was higher in the RTT patients but not statistically different (P>0.05). In the area of XCI≥65:35, the parental origin of X chromosome can be identified in 21 cases; among them the priority inactivated X chromosome was paternal origin in 18 cases (85.7%). The patients with highly skewed XCI (XCI≥80:20) had variable phenotypes.
Conclusions: XCI distribution pattern of the RTT patients was different from that of the RTT mothers and the control group, but non-random XCI was not significantly different among them. The transfer of the pathogenic gene of RTT mothers as carriers to the patients was not the main genetic pattern in RTT. In non-random XCI patients, the priority inactivated X chromosome was mainly of paternal origin. XCI could modify the clinical phenotype of RTT, but had limitations in explaining all the phenotypic manifestations of RTT.
X-linked mental retardation: searching for new genes and defining a new syndrome
Tian-Jian Chen, Yue-Ying Wang and Jose E. Martinez
Department of Medical Genetics, University of South Alabama, Mobile, AL, USA
Mental retardation (MR) is one of the most common and most complex disorders in clinical genetics and pediatric neurology. The prevalence of MR is about 2%-3% worldwide. X-linked mental retardation (XLMR) is the major cause for MR. XLMR is a highly heterogeneous genetic condition including over 140 syndromic and nonsyndromic disorders, such as Fragile X syndrome. In the recent three years, 15 genes have been identified to be associated with syndromic XLMR, and another 6 genes have been implicated in nonsyndromic XLMR. Identification of novel mutations on well defined XLMR genes results in a better understanding on genotype/phenotype correlation. Recently, we identified a novel mutation on RSK2 gene which has been associated with abnormal brain MRI in patients with Coffin-Lowry syndrome. We also defined a novel XLMR syndrome in a family with six affected males and two obligate carrier females in three generations. The first generation female carrier produced, from four different partners, four affected sons and a normal daughter. Her daughter went on to have two affected sons and two normal female offsprings, all from different fathers, strongly indicating an X-linked pattern of inheritance. All affected males have moderate mental retardation, early global developmental delay, microcephaly and craniofacial dysmorphism, short stature, ptosis, muscle atrophy, joint contractures, pigmentary abnormalities and hypogonadism. Clinical manifestations in this family presented a previously unreported XLMR. Cytogenetic studies of the male patients were negative, and Fragile X syndrome was ruled out by DNA analysis of the FMR1 gene. The defected gene for this condition was mapped in the region of Xp11-Xp22 by linkage analysis using STR markers. Research into the other nonsyndromic XLMR family, which included three affected males in two generations, the defected gene was mapped on Xq27.3 to Xq terminus. Mutation screening showed negative results in the XLMR gene in this region, including FMR1, FMR2 and MECP2. A novel XLMR gene was suggested. Further fine mapping and candidate gene search in the two regions are undergoing.
Genetic approach to dissecting the mechanisms of Fragile X mental retardation
Xin-Da Lin, Ai-Yu Yao, Fang-Fang Jiang, Dan Wang, Hui Zhi, Zhi-Hua Liu and Yong-Q Zhang
Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100080, China
Fragile X syndrome (FXS) is the most common form of inheritable mental retardation, with a prevalence of 1 in 4000 males and 1 in 8000 females. FXS is caused by the absence of the Fragile X mental retardation protein (FMRP) encoded by FMR1 resulting in the pruning defects of dendritic spines in the central brain. FMRP is an RNA binding protein, interacting with many other proteins to regulate the translation of its target mRNAs in neurons. However, many of its mRNA targets and interacting proteins, and the mechanism by which the absence of FMRP leads to mental retardation remain elusive. Taking advantage of the powerful Drosophila genetics, we established a Drosophila FXS model (Zhang et al, Cell 2001;107:591-603; Zhang and Broadie, Trends in Genetics 2005;21:37-45). We are now carrying out a large-scale genetic screening to isolate and characterize FMRP interacting genes. So far we have generated more than 70 Drosophila FMRP (dFMRP) suppressors. Preliminary analysis of the first batch of mutants showed that 15 suppressors have mutations within dFMR1, while 3 mutants are intergenic FMRP suppressors. Sequencing of genomic DNA from the mutants revealed that the mutations are located in different conserved domains of FMRP, which provide invaluable reagents to elucidate the structural and functional relationship of FMRP. Three intergenic dFMRP suppressors are being mapped and characterized to unravel the pathway in which FMRP participates. The systematic characterizations of both intragenic and intergenic dFMRP suppressors will be presented. Due to the high structural and functional homology between Drosophila FMRP and human FMRP, this study will significantly advance our mechanistic understanding of FMRP in the nervous system, which in turn will lead to the development of drugs for the disease.
Exploring a feasible method to diagnose mental retardation in the rural area of Shanxi province
Li-Wen Wang1, Xiao-Li Chen2, Liang-Ming Lin3, Xiu-Yuan Ding4, Ting Zhang2 and Jin Guo5
1) Department of Neurology, the Affiliated Children Hospital of Capital Institute of Pediatrics, Beijng, China; 2) Department of Molecular Immunology; 3) Department of Epidemiology; 4) Department of Medical Genetics, and 5) Department of Molecular Immunology, Capital Institute of Pediatrics, Beijng, China
Background: Mental retardation (MR), as one of the major birth defects, is a life-long human disability characterized by impairment of cognitive and adaptive skills. Due to its complex etiology, the national guidelines for evaluating MR are not yet available in China. In June 2004, with the help of neurologists of Capital Institute of Pediatrics, we diagnosed 173 children with mental retardation or development delay in Lvliang area in Shanxi Province. Among them only 7 patients (7/173, 4%) were clinically or genetically diagnosed with Down syndrome (DS). As to the remaining cases, how to identify the underlying causation of MR with the existed convenience there, is the biggest obstacle for further genetic research. In most of the rural regions of China the newborn screening has not been launched, and we also can not give overall EEG, EMG and intracranial imaging to every patient in the rural area. So the information about physical examination, laboratory tests, past medical history constituted our major evidence for diagnosis. Accordingly, looking for the subject with sporadic chromosome abnormality and familiar genetic mental retardation became our direction to strive.
Methods and Results: The criteria of recruitment are the following: either a family history of 2 or more patients with mental retardation or prominent facial feature, or accompanying 2 or more dysmorphic feature. With the criteria, 24 patients with familiar mental retardation and 60 patients with 2 and more malformation were identified. At the same time we found out that the ratio of male to female was 2.70:1 (the gender of 3 patients were unknown), which indicated X-linked mental retardation was the mainly pathogeny. Last year we selectively re-interviewed patients with syndrome or signs suspicious of genetic diseases in four villages of Lvliang area. A total of 15 patients underwent detailed genetic examination, questionnaire and cytogenetic analysis. This time we captured a family suspicious of Fragile X syndrome (2 children and 1 adult with MR) and a family suspicious of X-linked mental retardation (2 children and 4 adults with MR). Although the chromosome was normal, we will explore microdeletion or subtelomeric chromosomal rearrangements with fluorescence in situ hybridization (FISH). The molecular analysis of FMR1 gene will also be performed for confirmation of Fragile X syndrome.
Conclusions: This high primary evaluating yield of 27% (4/15) could be attributed to our screening method. It also suggested that in spite of lack of assistant examination, we paved a practical way for further study on other MR children in Shanxi province.
Pilot infant screening for Duchenne muscular dystrophy
C Yu, L Arthur, S Quary, B Coffee and PM Fernhoff
Division of Medical Genetics, Emory University, Atlanta, USA
Duchenne muscular dystrophy (DMD) and several other genetic disorders such as Fragile X syndrome, and sex chromosome aneuploidies do not fulfill the traditional newborn screening criteria. As technological methodology is available, newborn screening of these disorders and screening of older infants (6 through 12 months of age) open up another area to allow early detection of genetic disorders in general population to avoid "diagnostic odyssey" and to begin early intervention programs. Another benefit would be allowing ample time to provide genetic counseling and reproductive options before a family might have a subsequent at-risk pregnancy.
Emory Genetics and CDC are developing a pilot voluntary infant screening program in routine pediatric care for the early detection of DMD. Six to fifteen months old healthy male infants are recruited with informed consent to the study at their routine well check-up visit; males with DMD are recruited from DMA clinic at Children's Healthcare of Atlanta. A fluorometric assay method is developed to detect elevated creatine kinase (CK) levels in dried blood spots, and positive specimen was first analyzed with second tier CK isozymes assay on the same blood spot to rule out the false positive due to non muscle isozymes (primarily BB isozymes). Subsequently molecular confirmation on DBS was done using multiplex ligation-dependent probe amplification (MLPA) method to detect deletions and duplications of all of the 79 exons of the dystrophin gene.
A series of DBS samples enriched with known CK levels (0, 200, 400, 600, 800, 1000 U/L) were provided by the CDC Newborn Screening Quality Assurance Program for validation. A modified kinetic flurometric method was used for total CK assay using diadenosine pentaphosphate (DAP) to inhibit the endogenous adenylate kinase which may otherwise cause false positive results. The average percent recovery for the known spiked samples was 98.7% (96.9%-102.1%, n=31). The between-run CV was 9.66%-12.22% (n=31) and 3.92%-5.26% for within-run (n=24). The assay was linear up to approximately 2000 U/L. The preliminary normal range in healthy male was xxxx (n=50) and xxxx for DMD patients (n=10). Next we will validate CK isoenzyme assay using specific antibody and accumulate more data to generate cut-off level for CK screening before the pilot study implemented.
Transcriptome analysis of male embryonic gonads
Wai-Yee Chan1,2, Tin-Lap Lee1, Diana Alba1, Yun-Min Li3, Queenie P. Vong1, Vanessa Baxendale1, Owen M. Rennert1 and Yun-Fai Chris Lau3
1) Laboratory of Clinical Genomics, National Institute of Child Health and Human Development, NIH, Bethesda, Maryland, USA; 2) Department of Pediatrics, Georgetown University, Washington, DC, USA; and 3) Department of Medicine, VA Medical Center, University of California, San Francisco, CA, USA
Despite the identification of Sry, Wt1, Sf-1, Dax-1, Lhx9, Amh, and Sox9 as key genes during embryonic gonad development, the molecular networks controlling the earliest steps of male sex determination and subsequent events in gonad development is poorly defined. To understand the genes that regulate these processes, we employed Serial Analysis of Gene Expression (SAGE) to profile the transcriptome of mouse embryonic gonads at different embryonic times including embryonic day 10.5 (E10.5), E11.5, E12.5, E13.5, E15.5 and E17.5 days. Six high-quality SAGE libraries were constructed and sequenced with an average of 150 k coverage. A widespread bioinformatic analysis and comparison of SAGE transcriptomes of the developing gonad was performed to highlight the signature genes corresponding to the distinct changes during development through clustering and chromosome density mapping. 908 453 SAGE tags were sequenced identifying 70 489 different species of transcripts. Among these transcripts 4018 (5.7%) are novel, 21 130 (30.0%) are uncharacterized, and 45 341 (64.3%) are known genes. Hierarchical clustering dendrogram of 9748 tags having total counts of at least 4 in the six libraries clearly distinguished early events (E10.5, E11.5 and E12.5) from late events (E13.5, E15.5 and E.17.5) of gonad development. Importantly, each stage can be further subdivided. This provides the genomic fingerprint in defining gonad stages based on transcriptome activity. Chromosomal localization of expressed genes in the embryonic gonads revealed "hotspots" with significant changes at any time point. Mutations at any of these chromosomal clusters will presumably lead to abnormalities in gonad development. We are in the process of linking these hotspots to different mutation and functional annotation databases. Altogether, we have identified more than 70 000 genes in the six gonad libraries. Importantly, we observed a prominent increase in global genomic activity from E10.5 to E17.5, and identified important chromosomal regions related to the gonad developmental processes. These regions represent possible treatment targets and markers in early diagnosis for sexual developmental disorders in males.
Mechanism of mammalian sex determination
Yun-Fai Chris Lau, Yun-Min Li and Hyun Ju Oh
Department of Medicine, VA Medical Center, University of California, San Francisco, CA 94121, USA
The sex determining region Y (SRY) gene on the Y chromosome is the primary gene responsible for testis determination and differentiation during embryonic development, thereby switching on a male developmental pathway leading secondary sex characteristics. SRY is a DNA-binding transcription factor that interacts with other accessory factors to form a transcriptional complex(es). It is expressed only for short period during sex determination. It regulates the expression of downstream genes in the developmental process. Currently the exact nature of these accessory factors is uncertain. Using various protein interactive cloning strategies, we have identified two important proteins, the Krüppel-associated box only (KRAB-O) and the poly (ADP-ribose) polymerase (PARP-1), as putative SRY interactive proteins. Various molecular and cellular characterizations confirm the interactions and define the critical interactive domains of these factors with SRY. These studies have provided critical insights into the mechanism(s) by which SRY mediates its sex determination.
The gonadoblastoma gene on the Y chromosome
Yun-Fai Chris Lau, Yun-Min Li, Tatsuo Kido and Shane Oram
Department of Medicine, VA Medical Center University of California, San Francisco, CA 94121, USA
Failure in testis differentiation in the presence of a whole or portion of the Y chromosome predisposes XY sex-reversed individuals to gonadoblastoma at high frequency in early life. A locus on the Y chromosome, termed gonadoblastoma on the Y chromosome (GBY), is postulated to harbor a gene(s) that promotes tumor formation in the dysgenetic gonads. Recent complete sequencing of the human Y chromosome revealed that the testis-specific protein Y-encoded (TSPY) gene is currently the only functional and protein-coding gene at the critical region for GBY, thereby establishing this gene to be a strong candidate for this oncogenic locus. TSPY is a tandemly repeated gene that encodes a protein with significant homology to the oncoprotein, SET. TSPY binds to cyclin B and possibly plays a role in cell cycle regulation. Gonadoblastoma is relatively rare and develops almost exclusively in intersex patients. However, it shares significant morphological and pathological properties with testicular germ cell tumors (TGCTs), which is the number one cancer among young men at 14-34 years of age. TGCTs are hypothesized to arise from mutational events during the differentiation of primordial germ cells and gonocytes since many of the early germ cell markers, such as Oct-3/4, C-kit and placental alkaline phosphatase (PLAP), are expressed in TGCTs. Using various immunofluorescence techniques, we demonstrated that TSPY was expressed at early stages of germ cell development, similar to these early germ cell markers, during human embryogenesis. Further, TSPY was also co-expressed with these tumor germ cell markers in seven cases of gonadoblastoma. TSPY was first detected in minute foci of tumor precursor cells localized at the epithelial cell layer of dysfunctional and/or atretic follicles. TSPY persisted in the tumor cells that grew from these minute foci and gradually and completely filled up the atretic follicles. The tumors grew in size, but in confined morphology. At late stages, such confined growth could progress into a network of more loosely organized tumor cell clusters that, in turn, could take on more aggressive properties. Similar studies on the TSPY expression in TGCTs, seminoma and nonseminoma, demonstrated that TSPY is specifically expressed in the tumor germ cells of all seminoma, but not nonseminomas. Significantly, TSPY was expressed almost 100% in carcinoma in situ (CIS), the precursor for TGCTs. Over-expression of TSPY potentiated cell proliferation in culture and tumorigenicity in nude mice. TSPY mediated a rapid G2/M transition in the cell cycle of the host cells. Hence, its ectopic expression in the tumor germ cells suggests that it might play a critical role in the initiation and progression of gonadoblastoma and the more common cancer, TGCTs. Co-expression of TSPY with early germ cell markers in both gonadoblastoma and TGCTs supports a common etiological origin for both germ cell cancers.
Nationwide molecular epidemiologic investigation of hereditary hearing impairment in China
Xin-Hua Bao and Shi-Pu Dai
Genetic Testing Center for Deafness, Department of Otolaryngology Head Neck Surgery, Chinese PLA General Hospital, Beijing 100853, China
Approximately 1-2 in 1000 children are affected by severe or profound hearing loss at birth or during early childhood (prelingual deafness). In China, there are more than 20 million people suffered from the hearing impairment and more than 30 thousand children in 20 million births were born with severe-profound hearing loss every year. It is estimated that at least 50% of childhood profound hearing loss are attributed to genetic defects. This proportion is thought to be continually increasing as public health conditions improve, leading to a decrease in the prevalence of hearing loss resulting from viral or bacterial infections and administration of aminoglycoside.
We are now establishing a standard genetic testing lab for deafness and setting up a protocol for genetic screening and epidemiologic investigation for hereditary hearing loss in China. The data from our lab showed that the genetic background of hearing loss in China is as strong as that in Western countries, meanwhile, the common Chinese deafness genes and their mutation spectrums have their unique features. Among 2113 non-syndromic hearing impairment (NSHI) cases, 178 children were found to have homozygous GJB2 235delC mutation and 208 children have heterozygous GJB2 235delC mutation. GJB2 sequencing analysis of 1190 NSHI cases showed that 250 cases carried at least unambiguous pathogenic mutation, among them 98 cases carried homozygous 235delC, 299-300delAT, 176del16bp, 605 ins46bp mutations, 80 cases carried compound mutations and 54.26% of heterozygous GJB2 235 delC were accompanied by another mutation like 299-300delAT, 176 ins16bp, 35insG, 35delG, 155delTCTG, 313del14bp, etc. The comprehensive mutation and polymorphism spectrum of GJB2 gene was drawn according to the GJB2 sequence results of these 1190 cases from 18 provinces. Among 2993 deaf cases from 19 provinces, 96 deaf children were found to carry mitochondrial DNA A1555G, most of them have received the treatment of aminoglycoside antibiotics. Seventy-one family with 1555G positive proband were reviewed with family history in detail and 898 maternal family members from these 71 families with normal hearing benefit from the genetic screening for precaution of risk of aminoglycoside administration. Among the 61 deaf patients with enlarged vestibular aqueduct (EVA), 25 patients have homozygous mutations, 19 have compound mutations, 17 have heterozygous mutations in SLC26A4 gene. The data provide very strong evidence for that the EVA syndrome is genetic disease, and 75.4% EVA patients carried at least one IVS7-2 A>G mutation. In the 876 NSHI cases from 17 provinces, 132 cases carried at least one IVS7-2 A>G mutation (49 cases homo, 83 hetero), showing 15.1% Chinese NSHI patients may be related to deafness caused by IVS7-2 A>G mutation (mostly are EVA).
The significance of molecular screening for deafness in China is to provide evidences of prevalent contribution of hereditary hearing impairment. The outcome of this study suggests a new approach to preventing severe-profound NSHI by avoiding ototoxicity exposure in 1555G carriers and prenatal intervention in GJB2 and SLC26A4 deafness family.
Nuclear modifier gene for the phenotypic expression of deafness-associated mitochondrial 12S rRNA mutations
Min-Xin Guan
Division and Program in Human Genetics and Center for Hearing and Deafness Research, Cincinnati Children's Hospital Medical Center, and Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA; Zhejiang Provincial Key Laboratory of Medical Genetics, School of Life Sciences, Wenzhou Medical College, Wenzhou 325003, China
Human mitochondrial 12S rRNA A1555G mutation has been associated with aminoglycoside-induced and nonsyndromic deafness in many families worldwide. Our previous investigation revealed that the A1555G mutation is a primary factor underlying the development of deafness but is not sufficient to produce a deafness phenotype. However, nuclear modifier genes have been proposed to modulate the phenotypic manifestation of the A1555G mutation. Here we identified the nuclear modifier gene TRMU encoding a highly conserved mitochondrial protein related to tRNA modification. Genotyping analysis of TRMU in 613 subjects of an Arab-Israeli kindred and 210 Italian/Spanish families and 31 Chinese pedigrees carrying the A1555G or C1494T mutation revealed a missense mutation (G28T) altering an invariant amino-acid residue (A10S) in the evolutionarily conserved N-terminal region of Trmu protein. Interestingly, all eighteen Arab-Israeli/Italian/Spanish matrilineal relatives carrying both the TRMU A10S and 12S rRNA A1555G mutations exhibited congenital profound deafness. Functional analysis showed that this mutation did not affect importing of Trmu precursors into mitochondria. However, the homozygous A10S mutation leads to a marked failure in mitochondrial tRNA metabolisms, specifically reducing the steady-state levels of mitochondrial tRNAs. As a consequence, these defects contribute to the impairment of mitochondrial protein synthesis. Resultant biochemical defects aggravate the mitochondrial dysfunction associated with the A1555G mutation, exceeding the threshold for expressing the deafness phenotype. These findings indicate that the mutated TRMU, acting as a modifier factor, modulates the phenotypic manifestation of the deafness-associated 12S rRNA.
High resolution microarray comparative genomic hybridization reveals clinically relevant variations frequently occurring outside the known syndrome regions
Yao-Shan Fan
University of Miami Miller School of Medicine, Miami, FL, USA
Genomic imbalance is a major cause of developmental disorders. Chromosomal abnormalities are detected by conventional cytogenetic analysis in less than 5% of patients with unexplained mental retardation. Over 40 microdeletions syndrome regions have been described and several of them are routinely detected for clinical diagnosis by fluorescence in situ hybridization (FISH). Meanwhile, unbalanced chromosomal rearrangements involving the subtelomeric regions have been recognized as a significant cause of idiopathic mental retardation. A large number of data have shown that about 3% of patients with idiopathic mental retardation have a subtelomeric aberration. The technologies of microarray-based comparative genomic hybridization (array CGH) have been developed in recent years and a large number of large-scale variations in the human genome have been reported. BAC clone-based arrays targeting to the known microdeletion syndromes and the 41 subtelomeric regions (targeted array) have been used as a diagnostic tool. Array CGH for the whole genome has revealed clinically relevant variations in patients with unexplained mental retardation. We have established three array CGH platforms in our lab, i.e., a targeted array, a BAC clone based array for the whole genome with a ~1 Mb interval, and a high resolution oligonucleotide array containing 44 000 probes representing over 30 000 mapped human genes. Our results have demonstrated that clinically relevant large genomic alterations frequently occur outside the known syndrome regions. Our study strongly suggests that whole genome screening by a high resolution array CGH analysis can serve as a powerful diagnostic tool for unexplained developmental disorders.
Inborn errors of metabolism disease studies using tandem mass spectrometry
Xue-Fan Gu
Department of Pediatric Endocrinology and Genetic Metabolism, Xinhua Hospital, Shanghai Jiaotong University School Medicine, Shanghai Institute for Pediatric Research, Shanghai 200092, China
Tandem mass spectrometry has demonstrated the broad spectrum of disease covered, specificity and high sampling throughput. We report here the outcome study of 2110 cases suspected with inborn errors of metabolism collected in China from 2003 to December 2005, the amino acids and acycarnitine profiles were analyzed by API 2000 tandem mass spectrometry. The blood was collected on filter paper, punched and extracted into methanol solution with stable isotope labeled internal standards, then derivatized with butanolic-HCL and analyzed. Positive cases for the test were referred to the metabolic specialist who made a final diagnosis with other clinical and laboratory data. From the 2110 cases analyzed, 228 cases (10.8%) were given a final diagnosis. Among these cases, 156 were diagnosed as amino acid diseases (127 hyperphenylalaninemia, 10 ornithine transcarbamylase deficiency, 5 tyrosinemia type I, 5 maple syrup urine disease, 3 citrullinemia type I, 3 citrullinemia type II, 2 homocystinuria, 1 arginasemia); 63 cases were diagnosed as organic academia (32 methylmalonic acidemia, 10 propionic acidemia, 4 isovaleric acidemia, 3 glutaric acidemia type I, 5 3-methylcrotonyl CoA carboxylase deficiency, 2 3-hydroxy-3-methylglutaryl-CoA lyase deficiency, 3 biotinidase deficiency, 1 multiple CoA carboxylase deficiency, 3 b-keto thiolase deficiency); 9 cases were diagnosed as fatty acid disorders (4 medium chain acyl-CoA dehydrogenase deficiency, 1 carnitine palmitoyl transferase type II, 1 short chain acyl-CoA dehydrogenase deficiency, 1 very long chain acyl CoA dehydrogenase deficiency, 2 multiple acyl CoA dehydrogenase deficiency). Our results have demonstrated that the tandem mass spectrometry is an efficient and important tool in diagnosing amino acid diseases, organic acidemia and fatty acid oxidation disorders by measurement of one small punch of blood spot. The national network cooperation is the key step in services and further genetic and genomic study.
Association of progerin-interactive partners with lamina proteins
W. Ju2 and N. Zhong1,2
1) Peking University Center of Medical Genetics; and 2) New York State Institute for Basic Research, USA
The Hutchinson-Gilford progeria syndrome (HGPS or progeria) is a premature aging disorder of childhood. It results from a dominant negative effect of progerin, which is the truncated protein encoded by a mutant LMNA gene carrying G608G mutation. The detailed pathogenic mechanism of progerin involved in HGPS is yet unclear. To study this unknown mechanism, we have identified four novel progerin-interactive partner proteins (PIPs), including hnRNP E1, EGF, Mel18, and UBC9. These PIPs were found to have no differential interaction with normal lamin A/C when compared to progerin. To further investigate whether there is any abnormality that PIPs associate with lamina proteins, we performed co-immunoprecipitations in both normal and HGPS cells. Our results showed that (1) all PIPs but Mel18 associate with lamin B1, (2) all PIPs but UBC9 associate with emerin, (3) UBC9, EGF, and hnRNP E1 associate with SREBP, and (4) UBC9 and hnRNP E1 associate with LAP2a. The co-immunoprecipitations showed that UBC9 was able to pull down Mel18, EGF, and hnRNP E1; EGF pulled down UBC9 and hnRNP E1; and Mel18 pulled down hnRNP E1. Although our earlier results demonstrated that PIPs were co-immunoprecipitated with lamin A/C and progerin, the current study showed that progerin only pulls down hnRNP E1 but not EGF, nor UBC9. No significant difference could be noticed between normal and HGPS conditions. We also investigated if PIPs change their subcellular localization in the HGPS condition. Immunostaining of PIPs with nuclear markers including progerin showed that immuno-signals of all PIPs were within nuclei and there was no difference between normal and HGPS cells. Our results indicated that PIPs associate with lamina proteins; however, they are unlikely involved in the pathogenesis of HGPS.
The use of the primed in situ labeling technique in the detection of chromosomal subtelomeric region
Feng Li1, Run-Ming Jin1, Hui Yu1, Wen Li1 and Zhong Chen2
1) Department of Pediatrics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China, Jinrunm@public.wh.hb.cn; 2) Cytogenetics Laboratory, Division of Medical Genetics, Department of Pediatrics, University of Utah School of Medicine, Salt Lake City, UT 84132, USA, Zhong.Chen@hsc.utah.edu
Background: To establish a new, rapid and clinical practical molecular cytogenetic method for diagnosing the chromosomal subtelomeric deletion as well as guiding aristogenesis and prenatal diagnosis.
Methods: The primed in situ labeling (PRINS) reactions with chromosome 21 specific primer were performed on 30 peripheral lymphocyte samples of mental retardation children comparing with common chromosome karyotype analysis. By using tyramine signal amplification kit we carried out chromosomal subtelomeric PRINS labeling detection in the long arm of chromosome 4. We also performed PRINS examination in the long arm of chromosome 1 from 29 peripheral lymphocyte samples of mental retardation children by using tyramine signal amplification kit and mutil-group primers.
Results: The specific chromosomal signals were obtained through the PRINS method on both metaphase and interphase nuclei. In the samples of 21-trisomy disorder, an average labeling signals of 89% had displayed with 3 but in the samples of non-21-trisomy, an average labeling signals of 93% had displayed with 2. We set up successfully a chromosomal subtelomeric PRINS labeling technique in both long arms of chromosome 1 and chromosome 4 (1q and 4q). The experimental result of chromosomal subtelomeric PRINS labeling technique in the long arm of chromosome 1 was verified by flurescence in situ hybridization (FISH). In 29 samples of mental retardation, average labeling signals of 92% were displayed successfully but no deletion of long arm of chromosome 1 was found from the 29 mental retardation patients.
Conclusions: PRINS is a new, rapid and specific method to detect chromosome 1, 4 and 21 ameuploidy. PRINS provides the method of verification and complementary to traditional cytogenetics. The new technique is useful to clinical diagnosis of chromosomal diseases. We conclude that the microdeletion of long arm of chromosome 1 may be very rare in childhood mental retardation.
Effectiveness of methylation-specific multiplex ligation and probe amplification assay used for comprehensive molecular diagnosis of Angelman syndrome and Prader-Willi syndrome
David T. Miller1,2,3, Hong Shao1, Keith Tomaszewicz1, Virginia Kimonis2,4, Mira Irons2,4, Jan Schouten5 and Bai-Lin Wu1,4
1) Departments of Laboratory Medicine and Pathology, Children's Hospital Boston, MA, USA; 2) Division of Genetics, Children's Hospital Boston, MA, USA; 3) Division of Hematology, Brigham and Women's Hospital, MA, USA; 4) Harvard Medical School, Boston, MA, USA; 5) MRC Holland, Amsterdam, Netherlands, USA
Angelman syndrome (AS) is characterized by profound speech deficits, gait ataxia, seizures, characteristic EEG, microcephaly, and an unusually happy demeanor with propensity to paroxysms of laughter. The characteristic facial appearance includes a wide, smiling mouth with small, widely spaced teeth. Prader-Willi syndrome (PWS) is characterized by neonatal hypotonia, feeding problems, initial growth failure followed by childhood obesity, hypogonadism, behavioral problems and variable degrees of mental retardation, and minor dysmorphic features. AS and PWS, with very different phenotypes, have been of particular interest as both of them are imprinting disorders co-located at the same chromosome 15q11.2-13 (AS/PWS Critical Region), in which maternal and paternal copies of genes have differential function (i.e., the function of one copy of the genes in this critical region, either maternal or paternal, is essential). Thus, lack of function of the maternally inherited genes in this region, including UBE3A, is the underlying molecular deficit in AS because the paternally inherited copy is normally imprinted to prevent gene expression; disruptions of paternally inherited genes within the same region, including SNRPN, give rise to PWS because the maternally inherited copy is normally imprinted to prevent gene expression.
Clinical features of AS or PWS in the neonatal period may be subtle enough to delay diagnosis for the two neurodevelopmental disorders. At the same time, early molecular diagnosis is desirable because AS/PWS subjects may benefit from early initiation of appropriate therapy. Molecular diagnostic genetic testing can facilitate an early diagnosis of AS/PWS, as the majority of both have a diagnostic abnormality in parent-of-origin specific methylation within the AS/PWS Critical Region. With the DNA methylation assay, Angelman syndrome is diagnosed by indicating either a deletion of maternal DNA sequence (70%) or paternal uniparental disomy (UPD, 5%). The remaining AS cases are caused by the UBE3A mutations (10%-15%) or imprinting mutations involving maternal DNA sequences or other unknown mechanism; Prader-Willi syndrome is diagnosed by indicating either a deletion of paternal DNA sequence (70%) or maternal uniparental disomy (UPD, 25%), or imprinting mutation involving paternal DNA sequences (<5%).
Currently, most clinical laboratories use the Southern blotting to detect parent-of-origin methylation patterns in the AS/PWS critical region and use fluorescence in situ hybridization (FISH) to detect/confirm microdeletion, but the techniques require either large amounts of DNA or chromosome preparation; can not be automated; and is laborious and time-consuming. Multiplex ligation and probe amplification (MLPA) is a novel, simple, and cost-effective technique for DNA quantification, and has been applied to the detection of genomic deletions and duplications (also known as gene/sequences copy number changes) in a variety of syndromes, including PWS. The sensitivity of currently available MLPA in PWS is equivalent to FISH, demonstrated by our previous studies (Shao et al, 2005; Miller et al, 2006), because both tests only identify the cases caused by microdeletion which affected approximately 70% of patients with AS (maternal deletion) and 70% of patients with PWS (paternal deletion), respectively.
We have validated the effectiveness for a new modified MLPA technique that include detection of all possible abnormalities related to the parent-of-origin specific methylation at the AS/PWS Critical Region, so that the assay will have the ability to simultaneously detect deletions, duplications, UPD, and imprinting defects. By this approach the certainties for molecular diagnosis of AS and PWS will be up to 75% and 95% respectively in a single and rapid assay with a comparable sensitivity to that of the two separate tests (Southern blotting and FISH). To date, 9 out of 54 patients referred for genetic diagnosis of AS and 16 out of 78 patients referred for genetic diagnosis of PWS were found to have a methylation abnormality by the methylation-specific MLPA, which can be easily distinguished as a deletion, duplication or UPD. All the results were confirmed by Southern blot, FISH, and/or karyotyping analysis (as indicating of duplication) with 100% concordance. This report demonstrated that the methylation-specific MLPA is a comprehensive and effective testing strategy for early molecular diagnosis of Angelman syndrome and Prader-Willi syndrome.
Regional disease patterns of birth defects in China: a cohort study on the birth quality of the population
Xiao-Ying Zheng, Ting Zhang, Xin-Ming Song and Gong Chen
Population Based Studies on Birth Defect Prevention of Genetics and Environmental Determinants in China, Institute of Population Research/WHO Collaborating Center on Reproductive Health, The Center of Chinese Population Health and Development (CCPHD), Peking University, Capital Children Hospital, Beijing, China
Birth defects are major diseases affecting the health of the population. The causes of birth defects are complicated as involved with biochemical risk factors, socioeconomic and natural environmental risk factors. Birth defects as diseases with social characteristics should be studied within the context of human survival. The birth quality of population was studied in two counties of Shanxi Province, where the prevalence of birth defects has been the highest in China. The study included a sample survey and a cohort study on birth defects and intelligence development in a population of 230 000 people. In addition to demographic, social, economic and environmental characteristics, the survey also collected information from women with 20 weeks or more of pregnancy from January 1, 2002 to December 31, 2004. The information included their spontaneous abortions, termination of pregnancy for medical reasons, fetal deaths, still births and live births and the development of birth defects for infants and children under age 3 as well as the mother's general condition and nutritional status. Also collected were related geo-chemical, botanical, and biological samples of the living environment. The preliminary results showed that social, economic and environmental factors are very important in the development of birth defects. The prevalence of birth defects in the high-prevalence areas in Shanxi was 84.6‰. The main types of birth defects are neural tube defects, hernia, congenital hypophrenia, congenital heart disease, and congenital hydrocephalus, which accounted for 68% of the total birth defects. There were significant differences in the patterns of birth defects between the areas we studied and other countries and regions, suggesting that the risk factors leading to these abnormalities are specific in these areas. Among all birth defect cases identified, 21% were diagnosed during 20 to 27 weeks of pregnancy, 20% during 28 weeks of pregnancy to 7 days after birth, 28% during 7 days to less than 1 year, and 31% during 1 to 3 years of age. This distribution implies that only 20%-30% of birth defects can be detected by the current monitoring program in China. Spatial analysis showed that the occurrence of birth defects is clearly space-aggregated and is closely related to the socioeconomic and geographic environment, such as the types of soil. The nutrition condition of the population showed a serious imbalance in diets in reproductive women of child-bearing age with nutrition deficiency as the major characteristics. For example, the meat consumption of women of child-bearing age is accounting only for 6.7% of the national average. The intakes of retinol, calcium, folic acid, riboflavin, vitamin E and selenium are significantly lower. An analysis of metabolic products of folic acid and other related nutrients suggested that the lack or the deficiency of folic acid and vitamin B12 which are closely related to the metabolism of carbon metabolism unit and subsequent increase of homocysteine are closely correlated with the prevalence of neural tube defects. Based on these results, we hypothesize "the geography-nutrition-metabolic disorders leading to abnormality". Using both the macro- and microscopic approaches, this study first screened all the possible risk factors. This method will play an active role in exploring effectively the environmental risk factors of birth defects.
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