Pre-conception carrier screening for 21 inherited metabolic diseases in a Chinese population / 中华医学遗传学杂志

OBJECTIVE@#To determine the carrier rate for 21 inherited metabolic diseases among a Chinese population of childbearing age.@*METHODS@#A total of 897 unrelated healthy individuals (including 143 couples) were recruited, and DNA was extracted from their peripheral blood samples. Whole exome sequencing (WES) was carried out to screen potential variants among 54 genes associated with 21 inherited metabolic diseases. Pathogenic and likely pathogenic variants and unreported loss-of-function variants were analyzed.@*RESULTS@#One hundred fourty types of pathogenic/likely pathogenic variants (with an overall number of 183) and unreported loss-of-function variants were detected, which yield a frequency of 0.20 per capita. A husband and wife were both found to carry pathogenic variants of the SLC25A13 gene and have given birth to a healthy baby with the aid of preimplantation genetic diagnosis. The detected variants have involved 40 genes, with the most common ones including ATP7B, SLC25A13, PAH, CBS and MMACHC. Based on the Hardy-Weinberg equilibrium, the incidence of the 21 inherited metabolic diseases in the population was approximately 1/1100, with the five diseases with higher incidence including citrullinemia, methylmalonic acidemia, Wilson disease, glycogen storage disease, and phenylketonuria.@*CONCLUSION@#This study has preliminarily determined the carrier rate and incidence of 21 inherited metabolic diseases among a Chinese population of childbearing age, which has provided valuable information for the design of neonatal screening program for inherited metabolic diseases. Pre-conception carrier screening can provide an important measure for the prevention of transmission of Mendelian disorders in the population.

Analysis and prenatal diagnosis of FMR1 gene mutations among patients with unexplained mental retardation / 中华医学遗传学杂志

OBJECTIVE@#To analyze the (CGG)n repeats of FMR1 gene among patients with unexplained mental retardation.@*METHODS@#For 201 patients with unexplained mental retardation, the (CGG)n repeats of the FMR1 gene were analyzed by PCR and FragilEase@*RESULTS@#For the 201 patients with unexplained mental retardation, 15 were identified with full mutations of the FMR1 gene. The prevalence of fragile X syndrome (FXS) in patients with unexplained mental retardation was determined as 7.5% (15/201). Prenatal diagnosis was provided for 6 pregnant women with pre- or full mutations. Analysis revealed that women with mental retardation and full FMR1 mutations exhibited a skewed XCI pattern with primary expression of the X chromosome carrying the mutant allele.@*CONCLUSION@#FXS has a high incidence among patients with unexplained mental retardation. Analysis of FMR1 gene (CGG)n repeats in patients with unexplained mental retardation can facilitate genetic counseling and prenatal diagnosis for their families. FMR1 gene (CGG)n repeats screening should be recommended for patients with unexplained mental retardation.

Analysis of FMR1 gene CGG repeats among patients with diminished ovarian reserve / 中华医学遗传学杂志

OBJECTIVE@#To explore the correlation between Fragile X mental retardation gene-1 (FMR1) gene CGG repeats with diminished ovarian reserve (DOR).@*METHODS@#For 214 females diagnosed with DOR, DNA was extracted from peripheral blood samples. FMR1 gene CGG repeats were determined by PCR and capillary electrophoresis.@*RESULTS@#Three DOR patients were found to carry FMR1 premutations, and one patient was found to carry gray zone FMR1 repeats. After genetic counseling, one patient and the sister of another patient, both carrying FMR1 permutations, conceived naturally. Prenatal diagnosis showed that both fetuses have carried FMR1 permutations.@*CONCLUSION@#FMR1 gene permutation may be associated with DOR. Determination of FMR1 gene CGG repeats in DOR patients can provide a basis for genetic counseling and guidance for reproduction.

Genetic study on a consanguineous Chinese family with premature ovarian insufficiency caused by a missense mutation of PSMC3IP gene / 中国医师杂志

Objective:To explore the genetic etiology for a premature ovarian insufficiency (POI) patient from a consanguineous Chinese family, and to provide basis for genetic counseling and fertility counseling.Methods:Whole-exome sequencing was performed using DNA extracted from the blood sample of POI patient. Suspected pathogenic mutation was analyzed by bioinformatics methods and verified by Sanger sequencing. The pathogenicity of the variation was assessed according to the ACMG genetic variation classification criteria and guidelines.Results:A homozygous variation, c. 32G>T (p.G11V), of PSMC3IP was identified in the patient. Bioinformatics analysis revealed that the variation was conserved in different animal species, and this variation was classified as possible pathogenic variation according to the ACMG genetic variation classification criteria and guidelines.Conclusions:The homozygous missense variation of PSMC3IP is the cause of the POI patient in this family. We are reporting for the first time the missense variation in PSMC3IP gene caused POI, which enriched the mutation spectrum of PSMC3IP and provided the basis for genetic counseling and fertility guidance of this family.

Optimization of chromosome flaking technique for mesenchymal stem cells / 中华医学遗传学杂志

OBJECTIVE@#To optimize the condition for chromosome flaking of mesenchymal stem cells to ensure the cytogenetic quality control of expanding production and clinical application.@*METHODS@#Chromosomal flaking methods were optimized from current chromosome preparation techniques from the aspects of MSCs cell culture concentration, colchicine treatment time and low permeability time.@*RESULTS@#By repeated pre-experiments, the optimal MSCS chromosome flaking condition of MSCs was determined as cell culture concentration of (1-2)× 10 cells per T25 cell culture bottle, and the colchicines processing time was determined as 2 hours and 10 minutes, and the low permeability was 1 hour.@*CONCLUSION@#The optimized chromosome flaking condition can fulfill the requirement of cytogenetic quality control for MSCs.

Reproductive intervention for inborn errors of metabolism / 中华实用儿科临床杂志

Inborn errors of metabolism(IEM)are a large class of genetic diseases caused by congenital metabolic disturbance.IEM results in complex clinical phenotype and it is an important cause of disability,death,and stupidity for the children.The technique of reproductive intervention including prenatal diagnosis(PND)or preimplantation genetic diagnosis(PGD)is an important approach to avoid the birth of defect.According to the two basic characteristics of IEM,metabolic disturbance and gene mutation,the clinical application of PND/PGD technology in IEM are reviewed,and put forward a new idea for reproductive intervention based on carrier screening.

Karyotyping and analysis of 5α -reductase-2 gene mutation in 25 patients with hypospadias / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the karyotypes and SRD5A2 gene mutations in 25 patients with sporadic or familial hypospadias.</p><p><b>METHODS</b>The patients included 10 adults and 15 children, whose chromosomes were analyzed by G-banded karyotyping, and the SRD5A2 genes were sequenced.</p><p><b>RESULTS</b>Two patients were found to have an abnormal karyotype, while eight have carried compound heterozygous mutations of the SRD5A2 gene, which included 5 genotypes formed by 6 types of mutations, i.e., p.G203S/p.R227Q, p.R227Q/p.R246Q, p.Q6X/p.Q71X, p.L20P/p.G203S, and p.Q71X/p.R227Q. Mutations of the SRD5A2 gene were present in 32% (8/25) of all patients, 35% (8/23) in those with a normal karyotype, and 44.4% (8/18) in those with proximal type hypospadia. Bioinformatic analysis, literature review and pedigree analysis confirmed that all such mutations are pathogenic.</p><p><b>CONCLUSION</b>Chromosomal anomalies and mutations of the SRD5A2 gene are the main cause of hypospadias. Sequencing of the SRD5A2 gene may explain the etiology of nearly half of the patients with proximal type of hypospadas but a normal karyotype, which can facilitate genetic consulting.</p>

Study of two Chinese families affected with resistant ovarian syndrome resulted from novel mutations of FSHR gene / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the genetic etiology for two Chinese families affected with hypergonadotropic amenorrhea and normal number of antral follicles.</p><p><b>METHODS</b>Peripheral venous blood samples were collected from the families for the extraction of genomic DNA. Mutations of FSHR and LHCGR genes were screened using PCR and Sanger sequencing. Suspected pathogenic mutations were verified in other members of the families. Bioinformatics software and NCBI were used to analyze the pathogenicity of the mutations.</p><p><b>RESULTS</b>Two previously unreported homozygous mutations, c.419delA and c.1510C>T of the FSHR gene were found in the probands of family I and II, respectively. Pedigree and bioinformatics analysis suggested that both mutations were pathogenic. Literature review suggested that both families were affected with resistant ovary syndrome rather than premature ovarian failure.</p><p><b>CONCLUSION</b>Two novel mutations of the FSHR gene have been identified, which have enriched the spectrum of FSHR gene mutations and provided a basis for genetic counseling and direction for reproduction.</p>

Analysis of FOXL2 gene mutations in 5 families affected with blepharophimosis, ptosis and epicanthus inversus syndrome / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To screen for FOXL2 gene mutations in 6 patients with blepharophimosis, ptosis, and epicanthus inversus syndrome (BPES), and explore their genotype-phenotype correlation.</p><p><b>METHODS</b>Peripheral venous blood samples were collected from the patients for the extraction of genomic DNA. PCR and Sanger sequencing were employed to analyze the coding region and flanking sequences of the FOXL2 gene. Pathogenicity of the identified mutations was verified through literature review and bioinformatic analysis.</p><p><b>RESULTS</b>A heterozygous c.672_701dup30 mutation was found in the probands from the two familial cases, while three heterozygous mutations (two were novel), namely c.462_468del (p.Pro156Argfs*113), c.251T to A (p.Ile84Asn) and c.988_989insG (p.Ala330Glyfs*204) were detected in the three sporadic cases. Literature review and bioinformatic analysis indicated that all these mutations are pathogenic.</p><p><b>CONCLUSION</b>Identification of causative mutations in the BPES patients has provided a basis for genetic counseling and reproductive guidance. The novel mutations have enriched the mutation spectrum of the FOXL2 gene.</p>

Genetic analysis of three families affected with split-hand/split-foot malformation / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the genetic etiology of three families affected with split-hand/split-foot malformation (SHFM).</p><p><b>METHODS</b>Peripheral venous blood samples from 21 members of pedigree 1, 2 members of pedigree 2, and 2 members of pedigree 3 were collected. PCR-Sanger sequencing, microarray chip, fluorescence in situ hybridization (FISH), real-time PCR, and next-generation sequencing were employed to screen the mutations in the 3 families. The effect of the identified mutations on the finger (toe) abnormality were also explored.</p><p><b>RESULTS</b>Microarray and real-time PCR analysis has identified a duplication in all patients from pedigrees 1 and 3, which have spanned FKSG40, TLX1, LBX1, BTRC, POLL and FBXW4 (exons 6-9) and LBX1, BTRC, POLL and FBXW4 (exons 6-9) genes, respectively. A missense mutation of the TP63 gene, namely c.692A>G (p.Tyr231Cys), was found in two patients from pedigree 2. FISH analysis of chromosome 10 showed that the rearrangement could fita tandem duplication model. However, next-generation sequencing did not identify the breakpoint.</p><p><b>CONCLUSION</b>The genetic etiology for three families affected with SHFM have been identified, which has provideda basis for genetic counseling and guidance for reproduction.</p>

Study of a family with epidermolysis bullosa simplex resulting from a novel mutation of KRT14 gene / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To determine the molecular etiology for a Chinese pedigree affected with epidermolysis bullosa simplex (EBS).</p><p><b>METHODS</b>Target region sequencing using a hereditary epidermolysis bullosa capture array combined with Sanger sequencing and bioinformatics analysis were used. Mutation taster, PolyPhen-2, Provean, and SIFT software and NCBI online were employed to assess the pathogenicity and conservation of detected mutations. One hundred healthy unrelated individuals were used as controls.</p><p><b>RESULTS</b>Target region sequencing showed that the proband has carried a unreported heterozygous c.1234A>G (p.Ile412Val) mutation of the KRT14 gene, which was confirmed by Sanger sequencing in other 8 affected individuals but not among healthy members of the pedigree. Bioinformatics analysis indicated that the mutation is highly pathogenic. Remarkably, 3 members of the family (2 affected and 1 unaffected) have carried a heterozygous c.1237G>A (p.Ala413Thr) mutation of the KRT14 gene, which was collected in Human Gene Mutation Database (HGMD). Bioinformatics analysis indicated that the mutation may not be pathogenic. Both mutations were not detected among the 100 healthy controls.</p><p><b>CONCLUSION</b>The novel c.1234A>G(p.Ile412Val) mutation of the KRT14 gene is probably responsible for the disease, while c.1237G>A (p.Ala413Thr) mutation of KRT14 gene may be a polymorphism. Compared with Sanger sequencing, target region capture sequencing is more efficient and can significantly reduce the cost of genetic testing for EBS.</p>

Genetic analysis for 2 females carrying idic(Y)(p) and with sex development disorders / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the phenotype-genotype association of isodicentromere Y chromosome by analysis of two female patients carrying the chromosome with sexual development disorders.</p><p><b>METHODS</b>The karyotypes of the two patients were determined by application of conventional G banding of peripheral blood samples and fluorescence in situ hybridization (FISH). PCR was applied to detect the presence of SRY gene.</p><p><b>RESULTS</b>Conventional karyotype analysis showed case 1 to be a mosaic: mos.45,X[38]/46,X,+mar[151]/47,XY,+mar[5]/47,X,+mar × 2[2]/46,XY[4], FISH showed that 12 different cell lines were presented in the karyotype of case 1 and partial cell lines with SRY gene, the marker is an isodicentromere Y chromosome [idic(Y)(p)]. No mutation was found in the SRY gene. The karyotype of case 2 was mos.45,X[25]/46,X,+mar[35]. FISH showed the marker to be an idic(Y)(p) without the SRY gene.</p><p><b>CONCLUSION</b>The karyotype of patients carrying idic(Y)(p) seems unstable, and female patients have the characteristics of short stature and secondary sexual hypoplasia. Karyotype analysis combined with FISH analysis can accurately determine the breakpoint of idic(Y) and identify the types of complex mosaic, which may facilitate genetic counseling and prognosis.</p>

Abnormal expression of PEX10 gene may be related to epilepsy associated with 1p36 copy number variations / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To assess the association of PEX10 gene and 1p36 copy number variations in 1p36 region with concurrent epilepsy through analyzing 3 cases.</p><p><b>METHODS</b>The karyotypes of 3 patients were determined by high resolution chromosome banding, multiplex ligation dependent probe amplification (MLPA), fluorescence in situ hybridization (FISH) combined with single nucleotide polymorphism array (SNP) technology. Real-time PCR was carried out to determine the mRNA levels of PEX10 gene in peripheral blood of the patients.</p><p><b>RESULTS</b>No abnormality was found upon high resolution karyotyping. MLPA analysis showed that all of the 3 patients had a copy number variation of subtelomeric region in the short arm of chromosome 1, which was confirmed by FISH and SNP chip analyses. Case 1 and case 2 both had an epilepsy phenotype, and their copy number variations have encompassed the PEX10 gene. On the other hand, case 3 has absent epilepsy, and its PEX10 gene copy number was normal. Family investigation confirmed that the chromosome abnormalities in all of the 3 cases were of de novo type. Compared with healthy controls, real-time PCR showed that mRNA of the PEX10 gene was increased in case 1 but decreased in case 2.</p><p><b>CONCLUSION</b>The abnormal expression of PEX10 gene resulting from copy number variations of 1p36 region may be associated with the epilepsy phenotype.</p>

Study of a Bethlem myopathy pedigree resulted from a novel mutation of COL6A3 gene / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To determine the molecular etiology for a muscular dystrophy pedigree with target region sequencing platform using hereditary myopathy capture array.</p><p><b>METHODS</b>Specific gene testing was performed based on the clinical diagnosis. Since no pathogenic mutation was found, target region sequencing with hereditary myopathy capture array combined with Sanger sequencing and bioinformatics analysis were employed in turn. PolyPhen and NCBI were used to evaluate the pathogenicity of identified mutation and conservation of the gene.</p><p><b>RESULTS</b>Target region sequencing indicated the proband has carried a heterozygous c.3353 A>C mutation of COL6A3 gene, which was confirmed by Sanger-sequencing in 4 affected individuals from the family. The same mutation was not detected in healthy members of the pedigree. Bioinformatics analysis suggested that the mutation has caused a highly pathogenic amino acid substitution from Histidine to Proline. The affected patients featured normal intelligence with mild myogenic damage by muscle biopsy, slightly increased serum creatine kinase and slow disease progression, which was consistent with Bethlem myopathy.</p><p><b>CONCLUSION</b>Target region sequencing is an effective and efficient method for genetic testing. The heterozygous c.3353A>C mutation in exon 8 of the COL6A3 gene probably underlies the Bethlem myopathy with autosomal dominant inheritance.</p>

Analysis of a novel mutation of AR gene in a patient featuring mild androgen insensitivity syndrome / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the clinical and molecular genetics characteristics of a patient with mild androgen insensitivity syndrome (MAIS).</p><p><b>METHODS</b>Clinical data of the patient was collected, and DNA was isolated from peripheral blood sample. Eight exons of AR gene were amplified by PCR with specific primers and directly sequenced by Sanger method. The results were compared with standard sequences from GenBank. Online Polyphen-2 software was applied to predict the effect of mutation on the protein function and compare the conservation of the sequence at the mutation site in various species. The exon of the AR gene containing the mutated site was analyzed in 90 unrelated normal males using PCR and restrictive digestion with Sfa NI.</p><p><b>RESULTS</b>Sequence analysis has detected a novel missense mutation in codon 176 of exon 1 (Ser176Arg) of the AR gene. Analysis with polyphen-2 software has indicated the codon to be highly conserved across various species, and that the S176A mutation has caused damage to the protein structure and function (prediction score=0.999). The same mutation was not detected in 90 healthy males.</p><p><b>CONCLUSION</b>The S176A mutation of the AR gene may contribute to the mild androgen insensitivity syndrome.</p>

Chromosomal cryptic insertion of the terminal region and its formative mechanism determined by fluorescence in situ hybridization / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To determine the karyotype of a cryptic structural abnormality and explore the mechanism of apparent chromosomal terminal deletion.</p><p><b>METHODS</b>Fluorescence in situ hybridization(FISH) with a whole chromosome 7 painting probe and a 7q subterminal probe (7q36-->qter), prepared by chromosome microdissection technique, was used to analyze a case with a history of spontaneous abortion and 7q terminal deletion detected by conventional G-banding technique.</p><p><b>RESULTS</b>The case was a maternal cryptic insertional translocation between chromosome region 1p32 and 7q32-->q35. The region of chromosome 7q36-->qter was not inserted in to chromosome 1, and the abnormal chromosome 7 was not a terminal deletion but an interstitial deletion.</p><p><b>CONCLUSIONS</b>Chromosome insertion of the terminal region retains its telomere, which is consistent with the concept of a three-break rearrangement. Interstitial deletion may be regarded as another mechanism for terminal deletion in the chromosome banding level. Combined with chromosome microdissection, FISH technique could be a powerful diagnostic tool for detecting chromosome structural abnormalities.</p>

Advances in preimplantation genetic diagnosis for chromosomal translocation carrier / 中华医学遗传学杂志

Chromosomal translocation is a kind of common chromosomal abnormality. The carriers with chromosomal translocation could have more chance of normal pregnancy with the help of fluorescence in situ hybridization(FISH). This is a review aimed at analyzing the meiosis types of the translocation chromosome. The strategy of preimplantation genetic diagnosis for the carriers with chromosomal translocation is also discussed.