Genetic analysis of a pregnant woman with moderate intellectual disability due to variant of DLG4 gene / 中华医学遗传学杂志

OBJECTIVE@#To carry out genetic testing and prenatal diagnosis for a woman featuring moderate intellectual disability (ID).@*METHODS@#The patient had presented at the First Affiliated Hospital of Zhengzhou University on April 28, 2021. With informed consent, peripheral blood and amniotic fluid samples were collected for the extraction of genomic DNA. Pathogenic copy number variations (CNVs) were detected with CNV-seq, and single gene variants were detected by whole exome sequencing (WES) and Sanger sequencing. Candidate variant was verified by Sanger sequencing, and CNV-seq and multiplex ligation-dependent probe amplification (MLPA) were used to detect fetal CNVs.@*RESULTS@#The 23-year-old woman had moderate ID, sideway walking, and unstable holding. Ultrasonography at 18+3 weeks' gestation had revealed no fetal abnormality. No pathogenic CNV was detected in the woman by CNV-Seq, while WES revealed that she has harbored a heterozygous c.1675C>T (p.Arg559*) variant of the DLG4 gene, which was verified by Sanger sequencing. Based on guidelines from the American College of Medical Genetics and Genomics, the variant was predicted to be likely pathogenic (PVS1+PM2_supporting). Sanger sequencing has confirmed that the fetus has inherited this variant, and CNV-Seq also revealed that that fetus has harbored a 0.1 Mb heterozygous deletion at Xp21.1, which has encompassed the DMD gene, and the result was verified by MLPA.@*CONCLUSION@#The heterozygous c.1675C>T variant of the DLG4 gene probably underlay the mental retardation in this woman, and her fetus was found to harbor the same variant in addition with deletion of the DMD gene, which may predispose to ID type 62.

Analysis of genome copy number variations in fetuses with isolated ventricular septal defect and a literature review / 中华医学遗传学杂志

OBJECTIVE@#To assess the value of copy number variation sequencing (CNV-seq) for revealing the genetic etiology of fetuses with isolated ventricular septal defect (VSD).@*METHODS@#From December 2017 to December 2020, 69 fetuses with isolated VSD were identified at the First Affiliated Hospital of Zhengzhou University. Meanwhile, 839 similar prenatal cases were selected from public databases including Wanfang data, Wanfang Medicine, and China National Knowledge Infrastructure (CNKI) by using keywords such as "Ventricular septal defect", "Copy number variation", and "Prenatal". A total of 908 fetuses with isolated VSD were analyzed. CNV-seq was carried out for 69 fetuses.@*RESULTS@#Among the 908 fetuses, 33 (3.63%) were found to harbor pathogenic CNVs, which included 11 chromosomal aneuploidies (1.21%) and 22 pathogenic CNVs (2.42%). The pathogenic CNVs have involved 12 genetic syndromes, with those known to involve the heart development including 5 cases of 22q11.21 deletion syndrome, 2 cases of 4q terminal deletion syndrome, and 1 case of 9q subtelomere deletion syndrome. The outcome of pregnancies for 15 fetuses with pathogenic CNVs was known, of which 12 were terminated, and 3 had spontaneous closure of the ventricular septum after birth, but 1 of them had other abnormalities.@*CONCLUSION@#Fetuses with isolated VSD have a relatively high risk for chromosomal abnormalities, for which CNV-seq should be recommended.

Genetic diagnosis of non-classical 21-hydroxylase deficiency by the new nanopore sequencing detection method / 中华检验医学杂志

Objective:To summarize initial experience of applying nanopore third-generation sequencing detection method (nanopore sequencing) for genetic diagnosis of non-classical 21 hydroxylase deficiency (NC 21-OHD), and to explore its performance and application prospects.Methods:Clinical data of the two NC 21-OHD patients, who were hospitalized at the First Affiliated Hospital of Zhengzhou University in May 2019, were collected. Peripheral venous blood was collected and genome DNA extracted. Genetic variants was detected by nanopore sequencing and underwent bioinformatic analysis. Pathogenetic mutations in CYP21A2 gene were validated with PCR-sanger sequencing in the two patients and their parents.Results:The average reads length and sequence depth in the patient one was 12, 792 bp and 27.19×. The average reads length and sequence depth in the patient two was 13, 123 bp and 21.34×. Compound variants of c.293-13C>G/c.844G>T (p.Val282Leu) and c.332_339delGAGACTAC (p.Gly111Valfs)/c.844G>T (p.Val282Leu) were detected in these two patients, which were consistent with clinical phenotype of NC 21-OHD. Further analysis showed that c.293-13C>G mutation was inherited from her father and c.844G>T (p.Val282Leu) mutation was inherited from her mother for the patient one. The c.844G>T (p.Val282Leu) mutation was inherited from her father and c.332_339delGAGACTAC (p.Gly111Valfs) mutation from her mother.Conclusions:The heterozygous mutations in CYP21A2 gene are the cause of NC 21-OHD in these two patients. Nanopore sequencing technique is a reliable new detection method for patients with NC 21-OHD.

Clinical application of non-invasive prenatal diagnosis of phenylketonuria based on haplotypes via paired-end molecular tags and weighting algorithm.

BACKGROUND: Phenylketonuria (PKU) is a metabolic disease that can cause severe and irreversible brain damage without treatment. METHODS: Here we developed a non-invasive prenatal diagnosis (NIPD) technique based on haplotypes via paired-end molecular tags and weighting algorithm and applied it to the NIPD of PKU to evaluate its accuracy and feasibility in the early pregnancy. A custom-designed hybridization probes containing regions in phenylalanine hydroxylase (PAH) gene and its 1 Mb flanking region were used for target sequencing on genomic and maternal plasma DNA (7-13 weeks of gestation) to construct the parental haplotypes and the proband's haplotype. Fetal haplotype was then inferred combined with the parental haplotypes and the proband's haplotype. The presence of haplotypes linked to both the maternal and paternal mutant alleles indicated affected fetuses. The fetal genotypes were further validated by invasive prenatal diagnosis in a blinded fashion. RESULTS: This technique has been successfully applied in twenty-one cases. Six fetuses were diagnosed as patients carrying both of the mutated haplotypes inherited from their parents. Eleven fetuses were carriers of one heterozygous PAH variants, six of which were paternal and five of which were maternal. Four fetuses were absence of pathogenic alleles. All results were consistent with the prenatal diagnosis through amniotic fluid. CONCLUSIONS: The results showed that our new technique applied to the genotyping of fetuses with high risk for PKU achieves an accurate detection at an early stage of pregnancy with low fetal fraction in cell free DNA.

Genotype-phenotype and genetic analysis in five patients with Kallmann syndrome / 中华内分泌代谢杂志

To study the genotype-phenotype and genetic characteristics of Kallmann syndrome. Five patients with Kallmann syndrome were enrolled. Clinical data collection, chromosome karyotyping, whole exome sequencing (WES), and multiplex ligation-dependent probe amplification (MLPA) were used. All the five patients were males, aging from 2 months to 45 years old. Three of the five patients complained cryptorchidism, one complained gonadal dysgenesis, and one complained fasting hyperglycemia. The clinical feature was hypogonadotropic hypogonadism with anosmia, and all karyotype was 46 XY. Magnetic resonance imaging (MRI) showed undeveloped olfactory bulbs and tracts. Kallmann syndrome related gene novel variants were found in all the 5 patients. The hypoplasia of right kidney was found in a patient with c. 1795_1799del (p.Asn599Profs*66) of anosmin 1 (ANOS1) variant. Clinical heterogeneity and incomplete penetrance were seen in a patient with c. 2824A>G (p.Thr942Ala) of chromodomain helicase DNA binding protein 7 (CHD7). Besides, WES indicated a 109 bp-deletion on Xp22.31 (chrX: 8507699-8507804), which was the deletion of exon 10 on ANOS1 gene verified by MLPA. The deletion variant was inherited form his mother, and conformed to X-linked recessive inheritance. Kallmann syndrome is genetic and clinical heterogeneous. WES is helpful for early diagnosis. MLPA and genome copy number variation analysis (CNV) are also recommend if necessary.

Analysis of genetic variants in five pedigrees affected with Dysferlinopathy / 中华医学遗传学杂志

OBJECTIVE@#To analyze the clinical phenotype and genetic variants in five Chinese pedigrees affected with Dysferlinopathy.@*METHODS@#Next generation sequencing (NGS) was carried out for the probands from the five pedigrees. Suspected variants were validated by Sanger sequencing. Pathogenicity of the variants was assessed based on the standards and guidelines by the American College of Medical Genetics and Genomics (ACMG).@*RESULTS@#Ten DYSF gene variants (including 5 frameshift variants, 3 splicing variants, 1 missense variant and 1 nonsense variant) were detected. Among these, c.1375dupA (p.Met459Asnfs*15), c.610C>T (p.Arg204X), c.1180+5G>A and c.1284+2T>C were known to be pathogenic, while c.4008_4010delCCTinsAC (p.Leu1337Argfs*8), c.1137_1169del (p.379_390del), c.754A>G(p.Thr252Ala), c.1175_1176insGCAGAGTG (p.Met394Serfs*7), c.3114_3115insCGGC (p.Arg1040Profs*74) and c.1053+3G>C were unreported previously. Of the six novel variants, c.1137_1169del, c.1175_1176insGCAGAGTG and c.3114_3115insCGGC were predicted as pathogenic (PVS1+PM2+PM3), c.4008_4010delCCTinsAC as likely pathogenic (PVS1+PM2), c.754A>G and c.1053+3G>C as variants of uncertain significance based on the ACMG standards and guidelines.@*CONCLUSION@#Variants of the DYSF gene probably underlay Dysferlinopathy in the patients among the five pedigrees. Above finding has enriched the spectrum of DYSF gene variants.

Pathological variant of FBN2 gene identified in a pedigree affected with congenital contracture arachnodactyly / 中华医学遗传学杂志

OBJECTIVE@#To detect pathological variant in a Chinese pedigree affected with congenital contractural arachnodactyly (CCA).@*METHODS@#Next generation sequencing (NGS) was used to scan the whole exome of the proband. Potential variant of the FBN2 gene was also detected in all members of the pedigree and 100 healthy controls by Sanger sequencing. With the determination of the genotype, prenatal diagnosis was carried out by amniotic fluid sampling.@*RESULTS@#A c.3528C>A (p.Asn1176Lys) variant was identified in the FBN2 gene of the proband, other patients from this pedigree, as well as the fetus. The same variant was not found among healthy members from this pedigree and the 100 healthy controls.@*CONCLUSION@#The c.3528C>A (p.Asn1176Lys) variant of the FBN2 gene probably underlies the pathogenesis of CCA in our case. The new variant has enriched pathological spectrum of the FBN2 gene.

Clinical characteristics and variant analysis of five pedigrees with hereditary spastic paraplegia / 中华医学遗传学杂志

OBJECTIVE@#To explore the clinical and genetic characteristics of five pedigrees affected with hereditary spastic paraplegia(HSP).@*METHODS@#Clinical data of the five pedigrees was collected, and high-throughput sequencing was carried out to detect potential variants. Sanger sequencing were used to verify the results.@*RESULTS@#The probands of pedigree 1 and 2 were found to harbor heterozygous SPAST gene variants, namely c.1196C>T and c.1523T>A. The proband of pedigree 3 harbored compound heterozygous variants of FA2H gene (c.61G>C and c.688G>A). Proband from pedigree 4 harbored compound heterozygous variants of SPG11 gene (c.6812+4_6812+7delAGTA and c.915delT). The proband of pedigree 5 harbored compound heterozygous variants of SPG7 gene (c.1703_1704delAG and c.1937-1G>C). Based on the American College of Medical Genetics and Genomics(ACMG) guidelines, all variants were predicted to be likely pathogenic. Among these, SPAST gene c.1523T>A, FA2H gene c.61.G>C, SPG11 gene splicing region c.6812+4_6812+7delAGTA, c.915delT, SPG7 gene c.1703_1704delAG and splicing region c.1937-1G>C variants were unreported previously.@*CONCLUSION@#The probands of pedigrees 1 and 2 were diagnosed with autosomal dominant hereditary spastic paraplegia type 4, for which pedigree 2 showed incompletely penetrance. Pedigrees 3, 4, and 5 were diagnosed with autosomal recessive hereditary spastic paraplegia type 35, 11 and 7, respectively. Above result provided a reference for clinical diagnosis and genetic counseling for the affected pedigrees.

SGCA gene mutation analysis and prenatal diagnosis of two pedigrees with limb-girdle muscular dystrophy type 2D / 中华围产医学杂志

Objective:To analyze the variations of SGCA gene in two Chinese pedigrees of Han nationality with limb-girdle muscular dystrophy type 2D (LGMD2D) and provide prenatal diagnosis and genetic counseling for subsequent pregnancies within the pedigrees. Methods:This study involved two unrelated patients who were the probands of their pedigrees diagnosed with LGMD2D in the First Affiliated Hospital of Zhengzhou University from June 2017 to January 2018. Genomic DNA was extracted from peripheral blood samples of the probands and their parents. Coding sequences and flanking sequences of 21 LGMD-related genes from the probands were captured and subjected to high-throughput sequencing. Suspected mutations in their parents were detected and validated by Sanger sequencing and/or fluorescence quantitative polymerase chain reaction (PCR). Prenatal genetic diagnosis for high-risk fetuses in the two pedigrees was performed after the causative factors being identified.Results:(1) The proband of pedigree 1 carried compound heterozygous point mutations in SGCA gene with c.218C>G(p.P73R) and c.101G>A(p.R34H) inherited from his father and mother, respectively. Prenatal diagnosis indicated that the second fetus of the family carried the same mutations as the proband, and the family chose to terminate the gestation. (2) The proband of pedigree 2 inherited the compound heterozygous mutations of c.218C>T (p.P73L) and heterozygous deletion of exons 7 and 8 in SGCA gene from his parents. Their second fetus did not carry any of the above mutations and was delivered at full term. Serum creatinase level and physical, motor and mental development of the child were all within the normal range during a two-year follow-up after birth. Conclusions:The heterozygous mutations in SGCA gene are the cause of LGMD2D in the two pedigrees, and c.218C>G(p.P73R) and c.218C>T(p.P73L) are novel mutations. Genetic and prenatal diagnosis based on high-throughput targeted next-generation sequencing can rapidly and accurately detect the mutations responsible for LGMD2D.

Differential diagnosis of nonclassical 21-hydroxylase deficiency and polycystic ovary syndrome / 中华内分泌代谢杂志

Objective:To explore the differential diagnosis methods between nonclassical 21-hydroxylase deficiency(NC21-OHD) and polycystic ovary syndrome(PCOS).Methods:The clinical data of 31 women with NC21-OHD were compared with those of 29 women with PCOS.Results:Women with NC21-OHD showed a higher prevalence of adrenal hyperplasia and lower likelihood of polycystic ovary(PCO) than those with PCOS( P<0.05), with lower height( P<0.05). The levels of adrenocorticotropic hormone (ACTH), 17-hydroxyprogesterone(17-OHP), androstenedione(AD), total testosterone(TT), and progesterone were higher in women with NC21-OHD compared with those with PCOS( P<0.05). Women with PCOS had higher levels of luteinizing hormone(LH) and higher ratio of LH to follicle stimulating hormone(FSH) than those with NC21-OHD( P<0.05). The best two identification indexes for the two diseases were 17-OHP and progesterone, with the optimal cut-off points 3.34 ng/ml(sensitivity 89.7%, specificity 93.1%) and 0.64 ng/ml(sensitivity 90.0%, specificity 75.9%), respectively. During the 1-day mid-dose dexamethasone suppression test(DST), women with NC21-OHD had higher inhibition rates of 17-OHP, progesterone, AD, and TT( P<0.01) than those with PCOS. Their optimal cut-off values of suppression rates were 73.5%(sensitivity 95.2%, specificity 100.0%), 55.5%(sensitivity 100%, specificity 88.9%), 61.4%(sensitivity 84.2%, specificity 100.0%), 68.3%(sensitivity 65.0%, specificity 100.0%), respectively. Conclusion:The clinical manifestations of women with NC21-OHD are similar to those with PCOS. 17-OHP is the best differential indicator and the 1-day mid-dose DST plays an important role in the identification of the two diseases. Genetic analysis is the gold standard for distinguishing the two diseases.

Analysis of ETFDH gene variation in a Chinese family affected with lipid storage myopathy / 中华医学遗传学杂志

Objective@#To detect potential variation in an ethnic Han Chinese family affected with late-onset lipid storage myopathy.@*Methods@#Next generation sequencing (NGS) was used to screen disease-related genes in the proband. Suspected mutation was validated with PCR and Sanger sequencing in two patients, their father, and 100 healthy controls.@*Results@#Heterozygous c. 770A>G (p.Tyr257Cys) and c. 1395dupT (p.Gly466Tryfs) mutation were detected in the two patients. Their father was found to be heterozygous for the c. 770A>G (p.Tyr257Cys) mutation, while the c. 1395dupT (p.Gly466Tryfs) variation was not reported previously and not found among the healthy controls.@*Conclusion@#Mutations of the ETFDH gene probably underlie the pathogenesis in this family. The novel c. 1395dupT (p.Gly466Tryfs) has enriched the mutation spectrum of EDFDH gene.

Study on six patients with 17α-hydroxylase/17, 20-lyase deficiency / 中华内分泌代谢杂志

Objective@#To improve the understanding of 17α-hydroxylase/17, 20-lyase deficiency(17OHD)disease.@*Methods@#The clinical data of six patients suffering from 17OHD were analyzed retrospectively.@*Results@#Two patients with complete combined defect had typical clinical presentations, including absence of secondary sexual characteristics, primary amenorrhea, hypertension, hypokalamia, lower gonadal hormone levels, as well as elevated corticotropin and progesterone levels. TAC329AA homozygous mutation, IVS1+ 2T>C, and c. 775_776delAT complex heterozygous mutation were found in 2 cases. Four cases of partial combined defect showed high progesterone, lower gonadal hormones and dehydroepiandrosterone-sulfate levels. Three females(46, XX)showed spontaneous menstrual and primary infertility, and two of them got successful pregnancy with assisted reproductive technology. TAC329AA heterozygous mutation was found in those 4 cases.@*Conclusions@#TAC329AA mutation is common in 17OHD, and heterozygous or homozygous mutation of TAC329AA may be the genetic and molecular basis for determining whether these patients present as partial or complete defect. The elevated plasma progesterone in non-pregnancy combined with lower gonadal hormones and dehydroepiandrosterone-sulfate is the main character of patients with partial 17OHD. Less severe patients may be able to get successful pregnancy with assisted reproductive technology.

Study on six patients with 17α-hydroxylase/17,20-lyase deficiency / 中华内分泌代谢杂志

Objective To improve the understanding of 17α-hydroxylase/17, 20-lyase deficiency ( 17OHD ) disease. Methods The clinical data of six patients suffering from 17OHD were analyzed retrospectively. Results Two patients with complete combined defect had typical clinical presentation, including absence of secondary sexual characteristics, primary amenorrhea, hypertension, hypokalamia, lower gonadal hormone levels, as well as elevated corticotropin and progesterone levels. TAC329AA homozygous mutation,IVS1+2T＞C, and c.775 776delAT complex heterozygous mutation were found in 2 cases. Four cases of partial combined defect showed high progesterone, lower gonadal hormones and dehydroepiandrosterone-sulfate levels. Three females (46, XX) showed spontaneous menstrual and primary infertility, and two of them got successful pregnancy with assisted reproductive technology. TAC329AA heterozygous mutation was found in those 4 cases. Conclusions TAC329AA mutations are common in 17OHD, and heterozygous or homozygous mutation of TAC329AA may be the genetic and molecular basis for determining whether these patients present as partial or complete defect. The elevated plasma progesterone in non-pregnancy combined with lower gonadal hormones and dehydroepiandrosterone-sulfate is the main character of patients with partial 17OHD. Less severe patients may be able to get successful pregnancy with assisted reproductive technology.

Analysis of AR gene variant in an infant with complete androgen insensitivity syndrome / 中华医学遗传学杂志

OBJECTIVE@#To detect potential variant of AR gene in an infant with complete androgen insensitivity syndrome.@*METHODS@#The coding regions and splicing sites of the AR gene were subjected to PCR amplification and direct DNA sequencing. Fluorescence quantitative PCR was also used to detect copy number alterations of exons 2 to 8 of the AR gene.@*RESULTS@#Deletion of exons 2 to 8 was detected in the proband, and the results were verified among the family members.@*CONCLUSION@#Hemizygotic deletion of exons 2 to 8 of the AR gene probably underlies the complete androgen insensitivity syndrome in this infant.

Analysis of ETFDH gene variation in a Chinese family affected with lipid storage myopathy / 中华医学遗传学杂志

OBJECTIVE@#To detect potential variation in an ethnic Han Chinese family affected with late-onset lipid storage myopathy.@*METHODS@#Next generation sequencing (NGS) was used to screen disease-related genes in the proband. Suspected mutation was validated with PCR and Sanger sequencing in two patients, their father, and 100 healthy controls.@*RESULTS@#Heterozygous c.770A>G (p.Tyr257Cys) and c.1395dupT (p.Gly466Tryfs) mutation were detected in the two patients. Their father was found to be heterozygous for the c.770A>G (p.Tyr257Cys) mutation, while the c.1395dupT (p.Gly466Tryfs) variation was not reported previously and not found among the healthy controls.@*CONCLUSION@#Mutations of the ETFDH gene probably underlie the pathogenesis in this family. The novel c.1395dupT (p.Gly466Tryfs) has enriched the mutation spectrum of EDFDH gene.

Genetic screening and prenatal diagnosis in 18 high-risk families with 21-hydroxylase deficiency / 中华医学遗传学杂志

OBJECTIVE@#Genetic screening and prenatal diagnosis was performed in eighteen families with high risk of 21-hydroxylase deficiency (21-OHD) to provide valuable information for genetic counseling in these affected families.@*METHODS@#First, multiplex ligation-dependent probe amplification (MLPA) combined with nested-PCR based Sanger sequencing was used to detect CYP21A2 gene mutations in probands and their parents of eighteen families, with seven probands had been dead. Second, paternity test was applied to exclude the possibility of maternal genomic DNA contamination, and fetal prenatal diagnosis is based on the mutations found in proband or parents of the family.@*RESULTS@#Ten mutations were identified in these eighteen families, including large fragment deletion, I2G, E3del8bp, I172N, V281L, E6 cluster, L307Ffs, Q318X, R356W and R484Pfs. All probands were caused by homozygous or compound heterozygous mutations of CYP21A2 gene and their parents were carriers. By comparing short tandem repeat sites contamination of maternal genomic DNA was not found in fetal DNA. Prenatal diagnosis showed that five fetus were 21-OHD patients, four fetus were carriers and the other nine fetus were normal.@*CONCLUSION@#CYP21A2 gene mutation is the etiology of 21-OHD. Genetic testing of CYP21A2 could assist physicians in 21-OHD diagnosis and provided genetic counseling and prenatal diagnosis for parents who are at risk for having a child with congenital adrenal hyperplasia.

Regulation on phenotypic and functional maturation of dendritic cell by met-enkephalin / 中国免疫学杂志

Objective:To explore the mechanism of immunomodulatory effects of methionine-enkephalin(MENK)on dendritic cells(DC).Methods:We used scanning electronic microscope for DC morpholopy,assay for acid phosphatas activity,flow cytometry(FCM) and ELISA to study the effects of DC by MENK.Results:MENK(10-12mol/L) could increase the expression of MHC classⅡ,CD86 and CD40 molecules on DC surface(P