Understanding the application of genetic testing in practicing precision medicine for inherited ocular disease / 中华眼底病杂志

Inherited eye disease is a heterogeneous group of eye disorders caused by genetic defects, which has many genetic characteristics, such as multiple inheritance modes and numerous gene variation types. Over the past few decades, genetic testing has improved significantly, with more and more known diseasecausing gene variants identified. With the rapid development of high-throughput sequencing technology, clinical diagnosis and treatment of eye genetic diseases have been accelerated, and molecular diagnosis of eye genetic diseases has become an important step in accurate diagnosis and treatment. How to correctly select and evaluate each kind of genetic testing technology, reasonably standardize the use of genetic testing technology, and provide patients with more accurate genetic counseling are problem that clinicians need to seriously consider.

Cytogenetic and molecular genetic analysis of Klinefelter syndrome in a fetus of Duchenne muscular dystrophy family / 中华围产医学杂志

A 44-year-old pregnant woman (G5P3) who had delivered two children with DMD was admitted and underwent prenatal diagnosis at Peking Union Medical College Hospital in 2019. (1) The karyotype of the fetus in 2019 was 47,XXY. The fluorescence in situ hybridization (FISH) result showed a nucish(CSPX×2, CSPY×1)[100] and multiplex ligation-dependent probe amplification (MLPA) suggested sex chromosome abnormality. Based on the above results, the fetus was diagnosed with Klinefelter syndrome. Fetal short tandem repeat (STR) linkage analysis and Sanger sequencing indicated a heterozygous mutation of c.9543delG(p.Trp3181CysfsTer2). (2) Sanger sequencing of the proband found a novel frameshift mutation of c.9543delG(p.Trp3181CysfsTer2 ) in exon 65 of the DMD gene. (3) The male fetus performing prenatal diagnosis in 2008 was found to have the same maternal gene markers as the proband with the same genotype. While the genotype of the fetus in 2009 obtained a different maternal gene marker from the proband and did not detect the same DMD gene mutation. This fetus was delivered at full term and was good during follow-up. (4) The elder brother and cousin of the proband had the same frameshift mutation in exon 65 of the DMD gene as the proband. The mother of the proband was a heterozygous carrier of the mutation.

Pilot study of genetic counseling and prenatal diagnosis in androgen insensitivity syndrome: with 3 families reports / 中华妇产科杂志

Objective:To explore genetic counseling and prenatal diagnosis strategies for women who have androgen insensitivity syndrome (AIS) family history or pregnancy history of AIS proband.Methods:Three families of complete AIS (CAIS) were retrospectively reported and summarized. The subsequent pregnancies and processes of prenatal diagnosis were followed up.Results:Among three CAIS families, one family had androgen receptors (AR) gene mutation diagnosis; the other two families were diagnosed clinically without gene diagnosis. All three mothers of CAIS probands were in pregnant again when they sought counseling, with gestational weeks between 7－13 weeks. They underwent chorionic villi sampling or amniocentesis in their second trimester (at 12, 16, 17 weeks respectively). Chromosome gender of all three fetuses were 46,XY, which was inconsistent with the ultrasonographic phenotype of external genitalia. All patients chose selective abortion in their second trimester. The external genitalia of all aborted fetuses were female phenotype, which supported the diagnosis of CAIS.Conclusion:Genetic counseling and prenatal diagnosis should be provided to high-risk patients with family history of AIS or proband pregnancy history, so as to achieve the goal of good childbearing and sound childrearing.

The mutation analysis of PAH gene and prenatal diagnosis in classical phenylketonuria family / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To characterize the mutation spectrum of phenylalanine hydroxylase (PAH) gene and perform prenatal diagnosis for families with classical phenylketonuria.</p><p><b>METHODS</b>By stratified sequencing, mutations were detected in the exons and flaking introns of PAH gene of 44 families with classical phenylketonuria. 47 fetuses were diagnosed by combined sequencing with linkage analysis of three common short tandem repeats (STR) (PAH-STR, PAH-26 and PAH-32) in the PAH gene.</p><p><b>RESULTS</b>Thirty-one types of mutations were identified. A total of 84 mutations were identified in 88 alleles (95.45%), in which the most common mutation have been R243Q (21.59%), EX6-96A>G (6.82%), IVS4-1G>A (5.86%) and IVS7+2T>A (5.86%). Most mutations were found in exons 3, 5, 6, 7, 11 and 12. The polymorphism information content (PIC) of these three STR markers was 0.71 (PAH-STR), 0.48 (PAH-26) and 0.40 (PAH-32), respectively. Prenatal diagnosis was performed successfully with the combined method in 47 fetuses of 44 classical phenylketonuria families. Among them, 11 (23.4%) were diagnosed as affected, 24 (51.1%) as carriers, and 12 (25.5%) as unaffected.</p><p><b>CONCLUSION</b>Prenatal diagnosis can be achieved efficiently and accurately by stratified sequencing of PAH gene and linkage analysis of STR for classical phenylketonuria families.</p>

Microarray-captured next-generation sequencing as a diagnostic platform for detecting small mutations in Duchenne muscular dystrophy / 中华神经科杂志

Objective To set up a new diagnostic platform based on microarray exon-capture and next-generation sequencing for detecting small mutations in dystrophin gene.The sensitivity and specificity of the method were assessed in clinical settings and the distribution of small mutations in Chinese Duchenne muscular dystrophy/Becker muscular dystrophy (DMD/BMD) patients were also analyzed.Methods Forty-one DMD/BMD patients diagnosed by the clinical criteria without large deletion or duplication (≥ 1exon) were recruited from Peking Union Medical College Hospital consecutively.Genomic DNA was extracted from blood samples.The libraries were prepared.Then exon and intron-exon flanking sequences of DMD gene were captured by custom microarray.Targeted next-generation sequencing and Sanger Sequencing were conducted.The patients who were not detected any disease-causing mutation were performed muscle biopsy.Results Thirty-eight subjects were detected small mutations in DMD gene.All single nucleotide variants (SNVs) and insertion & deletions (INDELs) were validated by Sanger sequencing.Twenty-one novel mutations were reported.The distribution of SNVs and INDELs was similar to other international DMD databases.Upon immunohistochemistry staining of dystrophin protein,1 of 3 mutation-undetected patients was diagnosed as DMD,2 of them were excluded.The specificity of the method was 100％,while the sensitivity was 97.4％.Conclusions Our microarray-captured next-generation sequencing assay could detect SNVs and INDELs with high sensitivity and specificity.Its advantages are economic,time-saving and stable.The platform is suitable for clinical gene diagnosis.

Clinical study on 48 cases with complete 17α-hydroxylase deficiency / 中华妇产科杂志

Objective To investigate efficient diagnosis and treatment of 17α-hydroxylase (17OHD) deficiency by summarizing clinical characteristics of those patients.Methods From January 1983 to January 2010,48 cases with 17OHD in Peking Union Medical College Hospital were studied retrospectively.Results Among 48 patients with 17OHD,karyotype analysis showed,12 cases with 46,XX and 36 cases with 46,XY.The 46,XX karyotype and 46,XY karyotype with complete 17OHD had typical clinical presentation of amenorrhea [ 12/12,100％ ( 36/36 ) ],no typical spontaneous puberty [ 12/12,13.9％ (5/36) ],Hypertension [ 11/12,100％ ( 36/36 ) ],hypokalemia [ K +:( 2.6 ± 0.7 ),( 2.8 ± 0.7 )mmol/L],hypergonadotropin [ follicle-stimulatinghormone ( FSH ):( 51 ± 35 ),( 79 ± 46 ) U/L,luteinizing hormone( LH ):( 27 ± 14 ),(49 ± 37 ) U/L ],impaired production of sex hormones [ testosterone(T):0.003,0.005 nmol/L; estradiol ( E2 ):26.86,10.64 pmol/L ],hyper-progesterone [ (P):( 32 ± 15 ),( 29 ± 23) nmol/L],impaired production of 17α-hydroxyprogesterone ( 17α-OHP ) [ ( 2.5 ± 1.1 ),( 2.4 ±1.7) nmol/L],ACTH hypersecreation (91.8,114.0 pmol/L).ACTH stimulating test did not elevated in 17α-OHP and cortisol.Conclusion When patients with elevated basal serum levels of progesterone higher than that of ovulation period in addition to clinical symptoms,examination about 17OHD should be warranted.

Establishment of multiple quantitative fluorescent polymerase chain reaction assay and its application in rapid prenatal diagnosis of common chromosome aneuploidies / 中华妇产科杂志

Objective To establish the multiple quantitative fluorescent polymerase chain reaction (QF-PCR)assay and evaluate its clinical application in prenatal diagnosis.Methods Totally 170 samples Were collected between May 2008 and July 2009 in prenatal center of Peking Union Medical College Hospital:123 of them were amniotic fluid,9 were chofionic villous samples,20 were fetal blood and 18 were villi from aborted fetuses.All samples were from women of Han nationality,with mean age of (34.1±4.6) years old,and with mean gestational age of(19.6±1.0)weeks.Cytogenetic cultures and karyotyping were made to every sample.Genomic DNA wag extracted from the samples.The sequences of twenty short tandem repeat (STR) markers were designed according to the GenBank and references,including 6 STR markers in chromosome 21.4 in chromosome 18.4 in chromosome 13,4 in chromosome X,1 in chromosome Y and 1 universal marker in both X and Y chromosome.Each sample was amplified by two sets of multiple QF-PCR,which included 4 STR markers in each of 21,18,13 and sex chromosomes. If the result was uninformative,the third set of anotherd 4 STR markers was added. Results ( 1 ) Karyotyping. Cytogenetic analysis were made for all the 170 samples, 151 (89%) of which were normal, and 19 (11% ) were abnormal (2)QF-PCR assay. 167(98% ) samples were detected by QF-PCR. The results were obtained within 2 -3 days after sampling. 134 samples were proved normal by QF-PCR, which was consistent with karyotyping. Among the 19 abnormal karyotype samples, 18 were detected as abnormal( eight were 21-trisomy, three were 18-trisomy)by QF-PCR. Among the 167 samples, 150(90% ) were detected using the first and second set of STR mixtures, and 3(2% ) were detected when the third set of STR was added. The remain 14(8% ) were uninformative. (3) The diagnostic efficiency of QF-PCR. The sensitivity of QF-PCR in prenatal diagnosis of common aneuploidities was 95%, the specificity, the false positive rate, the false negative rate, the positive predictive value and negative predictive value were 100% ,0,5%, 100% and 99% , respectively. (4)Autusome and sex chromosome detection by QF-PCR. Among all the STR markers,D21S1270 and D21S1411 had the highest heterozygosifies in chromosome 21, and DXS8377 had the highest in sex chromosome. The amplifications were stable. Conclusion Multiple QF-PCR assay is a valid alternative in rapid prenatal diagnosis of common chromosome aneuploidies. With high accuracy, it can be used for numerous sample test in large-scale laboratories.

New multiplex-PCR assay for detection of deletions of DMD gene in Chinese DMD patients / 中华检验医学杂志

Objective To establish a new multiplex-PCR assay to improve the detection rate of mutations in the DMD gene in Chinese patients. Methods A retrospective review of DMD deletion spectrum of 355 DMD patients with deletions all over the gene was performed. All deletions were confirmed by " one-step approach" diagnostic procedure and MLPA analysis. The exons with high frequency of mutations were identified to constitute the amplification system and the PCR conditions were optimized. Results Two new multiplex-PCR assays were established. Assay one was used to detect 10 exons including exon 5, 8, 17, 44, 45, 47, 49, 50, 51 and 52 of DMD gene, in two PCR sets. The theoretical detection rate would be 92% (326/355). Assay two was used to detect 5 exons including exon 12, 19, 35, 43 and 54, which could be used to screen additional 5% (17/355) deletion cases. The method was validated in other 22 DMD patients. Multiplex-PCR results were completely identical to the MLPA results in all 22 DMD patients. Conclusions The two multiplex-PCR assays were established based on the analysis of 355 Chinese DMD patients with gene deletions. It is believed that the new approach would be more applicable for deletion detection on the Chinese DMD patients since the DMD cases involved were from the whole country.

Detection of DMD by Multiplex Ligation-depenclent Probe / 医学研究杂志

Objective Duchenne muscular dystrophy(DMD)is one of the most common X-linked recessive neuromuscular degeneration diseases.It is caused by genetic defects of dystropin gene with deletion,duplication,or point mutation that results in clinical muscle fatigue and dystrophy.Usually,gene deletion of one or a few exons of dystrophin accounts for about 55%～65% patients,duplication for about 5%～10% patients and point mutation for 25%.Most of hot-spot deletion mutation of DMD can be detected by multiplex PCR and the point mutation can be detected by PCR/sequencing analysis,however,it remains a challenge to detect duplication.The recently developed MLPA(multiplex ligation-dependent probe amplification)is an efficient procedure that can accurately analyze the copy number and deletion mutation of whole dystropin gene.Methods A validation for simultaneous detection of entire dystropin gene was performed with two reactions.Both of which detect 39 and half exons of dystrophin gene.Results Nine out of 15 patients with DMD were found to have deletion mutation in different exons of dystrophin gene.Among these 9 patients,7 were found having deletion previously with multiplex PCR for mutation of hot-spot by Peking Union Medical University.Two patients who had not been found deletion by multiplex PCR were shown to have rare deletion at exon 18 or 43 in this study.Conclusions MLPA provides a simple,rapid and accurate method of simultaneously detecting homozygous,heterozygous deletions and duplication mutation in two single reactions for all exons of dystrophin gene,which may be applied into clinical molecular analysis for DMD.

A Simplified Approach for Detecting Homologous Deletion of SMN1 Genes in Spinal Muacular Atrophy / 医学研究杂志

Objective To develop a rapid,reliable and convenient approach for diagnosing the homozygous deletion of SMN1 gene.Methods SMN1 gene was amplified specifically with double allele-specific PCR(AS-PCR).Meanwhile,one inrelevant gene was amplified as internal control by PAGE and agarose gel electrophoresis analysis to determine whether the sick children were with homozygous deletion of SMN1 genes.Results The homozygous deletion of exon7 in SMN1 gene was identified by agarose gel electrophoresis or PAGE accurately.Conclusion Compared to PCR-RFLP and DHPLC used in the past,this approach can diagnose homozygous deletion of SMA much more accurate,easier and more convenient without completed following analyses.