Investigation and analysis of genetic testing in a SMA cohort based on the national rare diseases registry system of China / 中华检验医学杂志

Objective:To explore application status and development trend of spinal muscular atrophy (SMA) genetic diagnosis technology based on the national rare diseases registry system of China.Method:A total of 200 SMA children registered at the Capital Institute of Pediatrics from July 2016 to December 2018 were included in this retrospective cross-sectional survey. The basic data, clinical subtypes, genotypes, and related genetic testing information of SMA children were obtained by checking SMA registration information, genetic testing reports, and also by telephone follow-up. The patient number and the composition of different genetic diagnosis technologies were analyzed by the stratification of genetic testing at various time. The correlation between the proportion of genetic diagnosis technology and genetic testing time was analyzed with Pearson correlation analysis.Result:There were 3 SMA cases with incomplete data, the remaining 197 SMA cases were included in this study. There were 37 (18.8%), 115 (58.4%) and 45 (22.8%) patients with type Ⅰ, Ⅱ and Ⅲ SMA, respectively. There were 185 cases of SMN1 homozygous deletion (93.9%), and 12 cases with compound heterozygotes (6.1%). Seven SMA-related genetic technologies were used from 2004 to 2017. MLPA accounted for 54.1% (100/185) used approach, followed by PCR-RFLP and first-generation sequencing, which accounted for 22.7% (42/185) and 10.3% (19/185), respectively. Nine, 6, 5 and 4 cases were tested with AS-PCR, qPCR, WES and DHPLC, respectively (2.2%-4.9%). The proportion of MLPA increased gradually since 2010 ( r=0.95, P<0.05), while PCR-RFLP declined gradually since 2004 ( r=-0.99, P<0.05). No correlation was found between technology and testing time for other genetic testing technologies ( P>0.05). The proportion of quantitative genetic technologies (MLPA, qPCR and DHPLC) increased gradually since 2010 ( r=0.94, P<0.05), and qualitative genetic technologies (PCR-RFLP, first-generation sequencing, AS-PCR and WES) decreased gradually since 2004 ( r=-0.94, P<0.05). The duplication detection rates of homozygous deletion and compound heterozygous mutation were 12.4% (23/185) and 41.7% (5/12), respectively (χ 2=5.86, P<0.05). During 2008-2015, the proportion of "the reports of both copy numbers of SMN1 gene and SMN2 gene" increased from 56.8% (21/37) in 2008-2015 to 69.1% (56/81) in 2016-2017. Conclusion:Genetic diagnosis of SMA has gradually developed from qualitative detection technology to quantitative detection technology, such as MLPA and qPCR, in China. In more and more SMA quantitative test reports, quantitative results of SMN2 gene are also provided in addition to quantitative results of SMN1 gene.

Antisense oligonucleotide therapies in neurodegenerative disease / 国际儿科学杂志

Antisense oligonucleotides(ASOs)are small sequences of DNA or RNA able to target mRNA transcripts resulting in change or reduction in the expression of target genes, and became an emerging class of therapeutic drugs.With its high specific, adaptable, and low side effects, ASO drugs are widely used in rare diseases, tumor, inflammatory diseases, cardiovascular disease, and infectious diseases.Recent numerous advancements in ASO sequence design, chemical modifications and delivery methods, significant breakthroughs have been made in the treatment and ASOs become an ideal candidate therapies for neurodegenerative diseases.

Clinical manifestations and genetics analysis of collagen type Ⅵ-related myopathy caused by variants in COL6A3 gene / 中华儿科杂志

Objective@#To summarize the clinical manifestations and determine the molecular etiology for two collagen type Ⅵ-related myopathy pedigrees.@*Methods@#Two spontaneous collagen type Ⅵ-related myopathy patients were admitted to Department of Neurology, Children′s Hospital, Capital Institute of Pediatrics in October 2017. Clinical data of probands and their family members were collected and their genomic DNA was obtained for genetic testing. Next generation sequencing was performed and the variants were verified by the Sanger sequencing in the family members.@*Results@#Target region sequencing indicated that the proband of family 1 has carried a heterozygous variant of COL6A3 gene, c.6229G>C(p.Gly2077Arg), and it was de novo variant confirmed by Sanger-sequencing in the family.The patient 1, a 2-year-three-month old boy, was admitted due to motor retardation at birth. He was defined as early severe Ullrich congenital muscular dystrophy. He never achieved independent ambulation, he had onset of symptoms was found at birth, including diffuse muscle weakness, striking distal joint hyperlaxity, proximal contractures, calcaneal protrusion, kyphosis, and hip dislocation. Serum CK level was elevated slightly and EMG showed neurogenic changes. The patient 2, a 7-year-old girl with a limp for 4 years, carried one de novo variant of COL6A3 gene,c.5169_5177del (p.Glu1724_Leu1726del). This variant results in the deletion of amino acids (1724 to 1726) in α3 chain of collagen Ⅵ, which may disturb the function of this protein.She was diagnosed as Bethlem myopathy with a mild phenotype. She had delayed motor milestones and presented with walking on tiptoe, hypotonia, and ithylordosis. The contracture of proximal joints was not very obvious. Serum CK level was normal and EMG showed myogenic changes.Muscle biopsy revealed muscular dystrophy and muscle magnetic resonance imaging of patient 2 showed vastus lateral is a "sandwich" sign. Immunofluorescence staining for COL6A3 chain in the cultured skin fibroblasts from patients 2 showed decreased deposition compared with control.@*Conclusions@#These two patients were diagnosed as spontaneous collagen type Ⅵ-related myopathy and carried different variants of COL6A3 gene. Different in pathogenetic variants could cause different genetic features and different phenotypes. Collagen type Ⅵ- related myopathy patients have various clinical manifestations. Typical phenotypes include muscular dystrophies, proximal contractures, and distal hyperlaxity. Muscle MRI shows diffuse fatty infiltration of gluteus maximus and thigh muscle. The histological staining showed the low level expression of COL6A3 chain. The seventy of phenotype was related to the genotype.

Screening for genetic mutations in hyperphenylalaninemia using Ion Torrent PGM sequencing / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a hyperphenylalaninemia related genes screening method using Ion Torrent Personal Genome Machine (PGM) for early detection and differential diagnosis of hyperphenylalaninemia (HPA).</p><p><b>METHODS</b>Three children with known HPA mutations and a healthy control were used for setting up the method. Ten children with HPA with known mutations were recruited for validating the method. Ion Ampliseq PCR was used to amplify the 5' and 3' untranslated region, coding sequence, and flanking introns of PAH, GCH1, PTS, QDPR, and PCBD1 genes. After the enrichment with the Ion OneTouch system, the products were sequenced by PGM. Data from the PGM were processed with Torrent Suite v2.2 software package. All variations were confirmed by Sanger sequencing.</p><p><b>RESULTS</b>For the 4 samples, the PGM output was 94.22 Mb, with approximately 99.5% of reads mapping to the target regions. Among these samples, we detected 74 variations (28 positions) including 6 known mutations. Compared with database and results of Sanger sequencing, 55 (18 positions) polymorphisms and 13 (4 positions) false positive calls were confirmed. For the 10 samples, all the known mutations were successfully identified.</p><p><b>CONCLUSION</b>Ion Torrent PGM sequencing is suitable for screening genetic mutation underlying HPA from the perspective of metabolic pathways, which can meet the clinical demand for individualized diagnosis and treatment.</p>

Distribution of common chromosomal karyotypes in patients with Turner syndrome and correlation between the mean age and height standard deviation scores on diagnosis / 中华实用儿科临床杂志

Objective To analyze the distribution of common chromosomal karyotypes of patients with Turner syndrome (TS), and to explore the correlation between the age and height standard deviation scores (HSDS) on diagnosis.Methods Retrospective investigation was performed for the data of age and HSDS on diagnosis in 273 TS girls(≤ 18.0 years old)diagnosed by chromosomal karyotypes.The main statistical methods were analyzed with t-test and Pearson correlation test by using the SPSS 18.0 statistical software.Results (1) There were 4 kinds of common chromosomal karyotypes in the TS :45, X (87/273 cases,31.9％),46, X, i (Xq) (43/273 cases, 15.7％) ,45, X/46, X, i (Xq) (36/273 cases, 13.2％) and 45, X/46, XX (23/273 cases, 8.4％), respectively, the adolescent TS all had delayed puberty.For the cases with 45, X karyotypes ,3 cases presented mental retardation and 2 cases with organs deformity.(2)The patients with 45 ,X/46,X,i(Xq) karyotypes or with 46,X,i(Xq) karyotypes had the maximum(12.56 age) or the minimum(9.70 age) mean age on diagnosis, respectively, there was a significant difference between 2 groups (t =3.019, P =0.004).The maximum deviation from normal height was found in the patients with karyotypes of 46, X,i (Xq) (HSDS =-4.04), and the minimum deviation was in the patients with karyotypes of 45,X/46, XX (HSDS =-3.16), and there was a significant difference between 2 groups (t =-2.95, P =0.004).(3) More than 75.7％ of TS patients was diagnosed when their heights deviated above 3 SD,and their mean age on diagnosis was 12.10 age,which was 3 years later than those patients within 2 SD.(4) There was a significant negative correlation between the age and HSDS on diagnosis in the groups of common chromosomal karyotypes[45,X、46,X,i(Xq) and 45,X/46,XX] (r =-0.551,-0.560,-0.622,all P ＜ 0.01), except for the group with the 45, X/46, X, i (Xq).Conclusions (1) In this study, the consti-tuent ratios of these 4 common chromosomal karyotypes were different from those in Europe and America's.(2)Patients with 45 ,X may have more severe symptoms than others.(3)The mean age on diagnosis was at least 3.0 years earlier when considered HSDS below-2.00 as an indicator for chromosomal karyotype screening,which would facilitate earlier diagnosis.

Identification of two survival motor neuron gene 1 gene mutations and evaluation of their effects on full-length survival motor neuron gene 1 transcripts / 中华神经科杂志

Objective To perform mutation analysis of survival motor neuron gene 1 (SMN1 in two spinal muscular atrophy (SMA) patients and their parents to evaluate the effects of the two SMN1 gene mutations on the transcript levels of the gene and preliminarily predict their effects on the structure and function of SMN protein.Methods Mutation analysis of SMN1 gene was carried out by multiplex ligationdependent probe amplification,reverse transcript-polymerase chain reaction (RT-PCR) and cloning sequencing.Transmission of the mutations was confirmed by the mutation analysis in patients' parents.The full-length SMN1 (SMN1-fl) transcript levels of the patients carrying these subtle mutations were detected using quantitative RT-PCR.Results The two patients were diagnosed as SMA Ⅱ and SMA Ⅲ.They carried p.Val19GlyfsX21 and p.Ala2Gly SMN1 mutations in SMN1 gene,respectively.Both of the two mutations were originated from their fathers.Compared with the healthy individuals (23.5 ± 4.9),the two patients had a significant reduction in the level of SMN1-fl transcripts (t =3.322,P =0.011 (p.Ala2Gly) ;t =6.964,P =0.000 (p.Val19GlyfsX21)).However,compared with the healthy carriers (14.1 ±4.5),the patient with p.Ala2Gly mutation had no significant reduction in the level of SMN1-fl transcripts (13.9 ±3.6,t =0.058,P =0.955) ; however,the patient with p.Val19GlyfsX21 mutation had a significant reduction (4.9± 2.4,t =3.725,P =0.004).Conclusions Two SMN1 gene mutations are identified in our study.The mutation p.Val19GlyfsX21 is a novel mutation and p.Ala2Gly is firstly reported in Chinese SMA patients.p.Val19GlyfsX21 may possibly lead to decreased SMN1-fl mRNA by nonsense-mediated messenger RNA decay,however,p.Ala2Gly has no obvious effects on the amount of the SMN1-fl transcripts,indicating that its deleterious effect may be occurring at SMN protein level or the function of SMN protein.

In vitro expression and structural analysis of four missense mutations (G247S, E280G, P362T, A434D) of phenylalanine hydroxylase gene / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To understand the pathogenic effect and the correlation between the genotype and phenotype of the 4 novel missense mutations (G247S, E280G, P362T and A434D) of phenylalanine hydroxylase gene (PAH).</p><p><b>METHODS</b>(1) The enzyme activity of the 4 mutants was assessed by using transient protein expression in mammalian cells. (2) The PAH amino acid sequences among different animal species were alignmented. (3) The effects of the 4 missense mutations on the protein structure were analyzed. (4) The clinical phenotype of the patients with PKU were analyzed, according to their blood Phe levels prior to treatment and the Phe tolerance.</p><p><b>RESULTS</b>(1) The residual enzyme activity expressed in vitro of G247S, E280G, P362T and A434D were 3.1%, 0.4%, 8.2% and 21.7% of the wild-type PAH respectively; (2)Gly247, Glu280 and Pro362 were among the highly conserved amino acids, while Ala434 was only moderately conserved; (3) As revealed by 3D structural analysis, G247S and E280G, being located at the active center of the enzyme, interfered with the binding of PAH to BH4 and ferrousion respectively, while P362T and A434D affected the formation and stability of the dimer and the tetramer of PAH; (4) As shown by clinical phenotypic analysis, classical PKU were observed in patients carrying G247S and E280G, moderate PKU were observed in patients carrying A434D, whereas both classical and moderate PKU were observed in patients carrying P362T.</p><p><b>CONCLUSION</b>(1) The E280G, G247S, P362T and A434D are all disease-causing mutations, with those located at the center of the enzyme displaying the most marked pathogenic effect; (2)The results of the structural analysis of the 3D molecule are consistent with the activity assessment of the enzyme expressed in vitro; (3) The consistency is observed between the genotype, the enzymatic activity expressed in vitro and the clinical phenotype.</p>

Construction and identification of nine single-point mutant recombinant plasmids of phenylalanine hydroxylase gene / 解放军医学杂志

Objective To perform PCR site-directed mutagenesis of nine novel PAH gene mutations (Y154H, R157I, Y206C, G247R, D282G, G346R, S349A, A389G, R400K) identified in northern Chinese and construct mutant recombinant plasmids of PAH gene. Methods 1) Every mutant recombinant plasmid was constructed according to the site of the mutation localized in functional domain of PAH gene and the related clinic phenotype of patients with the gene mutation. 2) Using the wild-type PAH expression vector as a templet, the mutant recombinant plasmids were directly amplified by PCR with Platinium Taq DNA polymerase and nine pairs of primers which were designed according to the human PAH cDNA sequence and the requirement for site-directed mutagenesis technology. 3) The positive strains were selected by Amp resistant test, PCR and restriction endonuclease analysis. The Mva Ⅰ, Mva Ⅰ, Hind Ⅲ, Rsa Ⅰ, Rsa Ⅰ sites exist in the sequences near the mutant sites of S349A, D282G, G247R, Y206C, Y154H, respectively, but not in the related sequence of wild-type PAH expression vector. Restriction endonuclease digestion could be directly used in identifying the mutant sites. However, the amplification created restriction site (ACRS) analysis was supplied in the followed identification of R157I, G346R, A389G, R400K. Finally the sequences of mutant recombinant plasmids of PAH gene were confirmed by DNA sequence analysis. Results Every sequence analysis showed that the mutant nucleic acids were introduced at the expected sites of PAH gene, suggesting that the mutant recombinant plasmids of PAH gene were constructed successfully. Conclusion PCR site-directed mutagenesis is accurate and highly efficient. The successfully mutagenized plasmids of PAH gene lay the foundation for the functional analysis of phenylalanine hydroxylase in mammalian cell system.