Incidental prenatal detection of an Xp deletion using an anonymous primer pair for fetal sexing

We report on the incidental prenatal detection of an interstitial X-chromosomal deletion in a male fetus and his mother by fetal sexing with a primer pair recognizing an X-Y homologous locus (DXYS19), formerly unassigned on the X chromosome. The proband asked for prenatal diagnosis because of her elevated age and risk of Duchenne muscular dystrophy (DMD). Prior to molecular genetic testing for DMD, fetal sexing was carried out on DNA prepared from cultured amniocytes. PCR analysis revealed the expected Y-chromosomal product, but did not show the constitutive X-chromosomal fragment. The absence of the X-chromosomal fragment in the fetus and on one X chromosome of the mother was confirmed by Southern hybridization of HindIII restricted DNA with probe pJA1165 (DXYS19). DXYS19X was mapped to Xp22.3 by combining several approaches, including: (1) analysis of somatic cell hybrid lines containing different fragments of the human X chromosome; (2) Southern hybridization of a yeast artificial chromosome (YAC)-filter panel provided by the Resource Center/Primary Database (RZPD); (3) FISH analysis; and (4) re-evaluation of two patients with interstitial deletions in Xp22.3. The extent of the deletion in the fetus was estimated by further markers from Xp22.3 and found to include the STS gene. Mental retardation could not be excluded since some mentally retarded patients exhibit overlapping deletions.     

Prenatal diagnosis of a familial Xq deletion in a female fetus: a case report

X chromosome deletion is an infrequent finding in prenatal diagnosis and presents a difficult counseling challenge when it occurs. We present a case of a familial X chromosome long arm deletion discovered in a routine amniocentesis and subsequently in the mother. The pregnancy resulted in the birth of a normal girl, and X chromosome inactivation skewing was demonstrated in both mother and daughter. Xq deletion phenotypes and counseling issues are reviewed.     

Prenatal diagnosis of DMD in a female foetus affected by Turner syndrome

OBJECTIVES: We report on a prenatal diagnosis of DMD complicated by a 45,X karyotype that was revealed only in the chorionic villus long-term culture. METHODS: Cytogenetic investigations were performed on both short-term (STC) and long-term cultures (LTC) of the chorionic villus sample. Familial segregation was performed using a panel of intragenic polymorphic markers, and multiplex PCR was used to characterize exonic deletion. RESULTS: Investigations performed for sex determination after STC of the chorionic villus sample showed a normal karyotype 46,XX, while the karyotype performed after LTC revealed a homogeneous monosomy X. Cytogenetic analysis performed on amniotic fluid cells showed 45,X/46,XX mosaicism. Familial segregation analysis for DMD showed loss of heterozygosity for the STR49 marker in the DNA of the proband, her mother and the foetus. Dystrophin gene analysis on the 45,X cells led to the identification of a deletion of exon 50. CONCLUSIONS: The report described a rare situation of monosomy X associated with a DMD genotype. The data confirmed the DMD carrier status of the proband and her mother and indicated that the foetus had a high risk to combine a Turner phenotype and DMD. This study illustrated the potential risk of using short-term culture of villi as the only source of biological material for prenatal diagnosis.     

Prenatal testing for a novel EBP missense mutation causing X-linked dominant chondrodysplasia punctata

OBJECTIVE: To determine the pathogenicity of a novel conserved missense mutation, p.Ser98Phe, in the emopamil binding protein (EBP) gene in order to perform a prenatal diagnostic test for X-linked dominant chondrodysplasia punctata (CDPX2) in a male foetus at 50% risk. METHODS: Family members were tested for p.Ser98Phe using PCR and sequence analysis of leucocyte or buccal cell DNA. Haplotype analysis was employed to identify the grandparental chromosome on which p.Ser98Phe had arisen. In silico protein analyses were used to predict whether p.Ser98Phe significantly altered the EBP protein structure. RESULTS: The mutation was detected in the proband and her affected mother but not in the maternal grandmother, who was thought to be mildly affected. However, haplotype analysis showed that p.Ser98Phe had arisen de novo on the grandpaternal X chromosome. Protein secondary structure predictions suggested that p.Ser98Phe alters the properties of the helix within which it is located. CONCLUSION: We concluded that p.Ser98Phe is likely to be pathogenic and proceeded with prenatal testing. The male foetus had not inherited p.Ser98Phe and his unaffected status was confirmed at birth. This family demonstrates some of the difficulties in interpreting the significance of novel missense mutations, particularly when results are needed urgently for prenatal diagnosis.     

Confirmation of a balanced chromosomal translocation using molecular techniques

Investigation of a couple, who had produced three babies with cri du chat syndrome, showed initially that the mother had an apparent deletion of chromosome 5. It seemed likely that she had a balanced chromosomal translocation but it proved impossible to detect the second chromosome involved using routine cytogenetic methods. Molecular techniques using quantitative hybridization dosage studies were performed and these showed that the mother had a double dose of DNA in the suspected deleted area of chromosome 5. Further studies, using in situ hybridization techniques, revealed that the missing segment of chromosome 5 had translocated onto the short arms of a C group chromosome and further analysis of prometaphase chromosomes showed the presence of a balanced translocation, 46,XY,t(5;9)(5qter----5p14.1::9p22----9pter;9 qter----9p22::5p14.1----5pter). As a result of these findings, it was possible to offer prenatal diagnosis to the patient in future pregnancies, by detecting the presence of a balanced or unbalanced translocation in the fetus using molecular and cytogenetic techniques.     

Fertility with deletion Xq25: report of three cases; possible exceptions for critical region hypothesis

We report on an Arab family in which a mother and two of her daughters, despite having deletion Xq25, are fertile. So far, only one case of deletion Xq25 associated with fertility has been reported. Consistent inactivation of the deleted X chromosome in the proposita and early menopause in the mother were noted. The effect of Xq deletion on fertility and the CRH is discussed.     

孕妇X染色体异常对其外周血游离DNA产前筛查的影响

目的探讨孕妇X染色体异常对于其外周血游离DNA(cf-DNA)产前筛查的影响。方法收集2016年4月1日至2019年5月31日于中国医学科学院北京协和医院就诊且cf-DNA产前筛查提示胎儿性染色体非整倍体异常(SCA)高风险的孕妇共124例,遗传咨询后行侵入性产前诊断。对于侵入性产前诊断结果与cf-DNA产前筛查结果不相符者,取孕妇白细胞、提取孕妇DNA进行大规模平行测序,以检测孕妇X染色体是否存在数量异常或拷贝数变异。结果124例cf-DNA产前筛查提示胎儿SCA高风险的孕妇中,除9例拒绝诊断,余115例均行侵入性产前诊断,其中41例与cf-DNA产前筛查结果相符,74例不相符。在结果不相符的74例孕妇中,孕妇DNA大规模平行测序发现孕妇X染色体数目异常或携带拷贝数变异者19例,占SCA假阳性孕妇的比例为25.7%(19/74),占总SCA高风险病例的15.3%(19/124)。结论孕妇X染色体数目异常、嵌合或携带拷贝数变异会影响cf-DNA产前筛查的结果,导致假阳性或假阴性结果;对于cf-DNA产前筛查提示胎儿SCA高风险的孕妇,应强调该结果可能受孕妇X染色体异常的影响。对于侵入性产前诊断和cf-DNA产前筛查结果不一致的孕妇,推荐对孕妇染色体进行检测,以明确假阳性或假阴性的原因;而对已明确X染色体数目异常或携带拷贝数变异的孕妇,则不推荐进行cf-DNA产前筛查。     

Familial X centromere variant resulting in false-positive prenatal diagnosis of monosomy X by interphase FISH.

Interphase fluorescent in situ hybridization (FISH) analysis performed on uncultured amniotic fluid cells from a female fetus revealed a single signal using an X chromosome alpha-satellite probe, and the absence of any signal using a Y chromosome alpha-satellite probe. This result was initially interpreted as monosomy for the X chromosome in the fetus. Subsequent chromosome analysis from the cultured amniotic fluid cells showed two apparently normal X chromosomes. FISH using the X alpha-satellite probe on metaphase spreads revealed hybridization to both X chromosomes, although one signal was markedly reduced compared to the other. The same hybridization pattern was observed in the mother of the fetus. This is the first report of a rare familial X centromere variant resulting in a false-positive diagnosis of monosomy X by interphase FISH analysis for prenatal diagnosis.     

Detection of a novel dystrophin gene mutation through carrier analysis performed during prenatal diagnosis in a case with intragenic recombination

OBJECTIVES: To report a multi-technical approach to Duchenne muscular dystrophy (DMD) mutation testing through carrier analysis, in the prenatal diagnosis of a male foetus without a known mutation segregating in the family and with inconclusive results of linkage analysis. METHODS: Haplotype analysis with the DMD region markers for assigning the carrier status of the mother and for prenatal diagnosis of foetal DNA; semiquantitative multiplex analysis of maternal and foetal DNA for the promoter and for 34 exons of the DMD gene; sequencing analysis of the maternal and foetal DNA for confirmation of the results. RESULTS: Because of an intragenic recombination of the DMD gene in foetal DNA, haplotype analysis gave inconclusive results. Semiquantitative PCR analysis displayed a pattern compatible with a heterozygous exon 60 mutation in the mother's DNA, while foetal DNA showed a normal migration pattern. Sequencing analysis confirmed the presence of a novel 7 base-pair deletion in exon 60 of the DMD gene in the mother and excluded the deletion in the foetus. CONCLUSION: Semiquantitative PCR results allowed the DMD mutation detection in the mother and the exclusion in the foetus, showing its crucial importance in prenatal diagnosis in those cases where linkage analysis is not conclusive.     

应用荧光原位杂交技术诊断羊水细胞染色体异常

探讨荧光源位杂交技术于产前诊断羊水细胞染色体异常的实验方法的应用价值。方法对３４例孕１６－２３周有产前认旨征者经腹部抽取羊水,用Ｃｈａｎｇ培养液传代培养,常规制备羊水细胞分裂中期染色体,应用生物素及地高辛标记的人类全着线粒探针和Ｘ、Ｙ、１３、２１、１８号染色体     

Prenatal diagnosis of inherited satellited non-acrocentric chromosomes

We report on the prenatal diagnosis of two sib female fetuses with a satellited short arm of chromosome 4 and a male fetus with a satellited long arm of chromosome X. The first two fetuses had a cryptic balanced translocation (4;15)(p16;p11.1) inherited from a mother carrying a satellited 4p and having an affected child with the Wolf-Hirschhorn syndrome. The third fetus had a satellited Xq, with a deletion of subtelomeric region of Xq. The mother was subsequently found to have the same satellited Xq but without the presence of a reciprocal translocation. She decided to continue the pregnancy. The proband with a satellited Xq manifested developmental delay, mental retardation, hypertelorism, ptosis of one eye, low-set ears, and hearing disturbance at age 6 months. Fluorescence in situ hybridization (FISH) with a specific telomeric or subtelomeric probe, and genetic marker analyses were used to confirm the diagnosis. Pregnant women with satellited non-acrocentric chromosomes are at risk for carrying fetuses with chromosome abnormalities. If the X chromosome is involved, the fetuses can be affected with X-linked recessive disorders including mental retardation. Detailed genetic counselling, cytogenetic studies, FISH and genetic marker analyses are useful in prenatal detection of abnormal chromosome rearrangements.     

Prenatal diagnosis and molecular cytogenetic characterization of a small supernumerary marker chromosome (sSMC) inherited from her mosaic sSMC(15) mother and a literature review

ObjectiveWe characterized a maternally inherited small supernumerary marker chromosome (sSMC) derived from chromosome 15 according to prenatal detection and made a review on the prenatal sSMC(15) cases with mosaic maternal inheritance.Case reportA 29-year-old woman underwent amniocentesis at 19 weeks of gestation due to the high risk of Down syndrome in maternal serum screening. No abnormalities were observed in prenatal ultrasound findings. G-banding analysis revealed a karyotype of 47,XX,+mar. Subsequently, we recalled the couple back for chromosomal analysis. The father's karyotype was normal while the mother's karyotype was 47,XX,+mar[15]/46,XX[35]. Molecular genetic analysis was utilized to identify the marker chromosome. The chromosomal microarray analysis (CMA) results of the mother showed there existed microduplications in the locus of 14q32.33, 15q21.1, 19p12 and Xq26.2, respectively. Then Fluorescence in situ hybridization (FISH) using specific probes for chromosomes 13/21, 14/22, and 15 was applied on the mother and the fetus. And the marker chromosomes for the mother and the fetus were all finally identified as inv dup(15) (D15Z1++, SNRPN-, PML-), which illustrated that the fetus inherited the sSMC(15) from her mother. Finally, a healthy female infant was delivered with no phenotypic abnormalities at 39 weeks.ConclusionThe combined utilization of the molecular genetic technologies, such as FISH and CMA, plays a critical role in the identification of the origins and genetic constitutions of sSMC, which would make a significant contribution to genetic counseling and prenatal diagnosis.     

A pregnancy with discordant fetal and placental chromosome 18 aneuploidies revealed by invasive and noninvasive prenatal diagnosis

This study investigated a pregnancy where the fetus was diagnosed with monosomy 18p by invasive amniocentesis and karyotyping. Additional noninvasive prenatal diagnosis, which detects fetal chromosome abnormalities in the circulating cell-free plasma DNA originating from the placenta revealed a related 18p monosomy/18q trisomy, suggesting confined placental mosaicism. Based on recent observations of chromosomal instability in the early preimplantation embryo, this study speculates on the possible embryonic origin(s) of these related but discordant chromosome 18 aneuploidies in the placental and fetal tissues. The findings highlight the potential for both false-positive and -negative noninvasive prenatal diagnosis results in pregnancies where there is either confined placental mosaicism or placental mosaicism.The study investigated a pregnancy involving a fetus with a chromosome disease syndrome called monosomy 18p where part of the short arm of chromosome 18 was missing in the fetal tissues. Using non-invasive prenatal diagnosis which detects fetal chromosome abnormalities in the circulating cell free plasma DNA originating from the placenta, we also detected monosomy 18p as well a related chromosome 18 abnormality involving duplication of the long arm of chromosome 18. This suggested confined placental mosaicism where the constitution of the chromosomes are different between fetal and placental tissues. We speculated that these related chromosome 18 abnormalities arose during preimplantation embryo development, leading to the formation of different chromosome abnormalities observed in the placental and fetal tissues of this pregnancy. Our findings highlight the potential for both false positive and negative non-invasive prenatal diagnosis test results in pregnancies where there is confined placental mosaicism.     

Prenatal diagnosis of mos46,X,del(Y)(q11.2)/45,X by cytogenetic and molecular studies with multiplex STR analysis

OBJECTIVES: To present the prenatal diagnosis of a fetus of mos46,X,del(Y)(q11.2)/45,X by cytogenetic and molecular analysis. CASE AND METHODS: A 35-year-old pregnant woman came to our hospital for amniocentesis, and fetal chromosomal aberrations with mos46,X, + mar/45,X were found. Fluorescence in situ hybridization revealed the existence of a Y centromere on the marker chromosome. Analysis with six pairs of short tandem repeat markers showed that the genomic DNA extracted from the uncultured amniotic fluid cells contained a deletion of Yq11.1-Yq11.2. Spermatogenesis loci of the Y chromosome were studied using four sets of multiplex PCR. The proximal two markers DYS271 and KALY were present and the other 16 distal markers were deleted. No deletion was noted in the Y chromosome of the father. RESULTS: Cytogenetic and molecular analyses revealed deletions of AZFb, d, and c regions on Yq11.2-Yqter in the fetal Y chromosome. Postmortem examination of the fetus showed a grossly normal male fetus with normal external genitalia and testes. CONCLUSION: The present report demonstrates that molecular analysis using polymorphic microsatellite markers and multiplex PCR is a useful complement to cytogenetic methods for the identification and the characterization of Y-chromosomal deletions.     

Prenatal diagnosis of factor X deficiency using a combination of direct mutation detection and linkage analysis with an intragenic single nucleotide polymorphism

OBJECTIVE: To present a family in which it was possible to perform prenatal diagnosis for recessively inherited factor X deficiency using both direct mutation detection as well as linkage analysis. METHODS: In a family where both parents were known to be carriers of factor X deficiency, fetal DNA was obtained from an ongoing pregnancy by CVS in the first trimester. Direct DNA sequencing was used to detect a previously identified factor X mutation, and linkage analysis using a single nucleotide polymorphism (SNP) was used to follow the segregation of the other parent's factor X alleles. RESULTS: Our studies predicted the fetus in question to be a heterozygote carrier of factor X deficiency, and this was demonstrated to be correct at birth. CONCLUSIONS: This is the first reported case of prenatal diagnosis of factor X deficiency. In addition, this case demonstrates the remarkable utility of SNPs in linkage analysis of rare genetic disorders.     

Prenatal diagnosis of autosomal dominant hereditary spastic paraplegia (SPG4) using direct mutation detection

OBJECTIVE: To present a report on prenatal diagnosis using direct SPG4 gene analysis in a family with autosomal dominant hereditary spastic paraplegia (AD-HSP). METHODS: Genetic linkage and haplotype analysis were previously carried out with chromosome 2p markers. DNA was obtained from affected individuals, the affected father, the mother, and fetal DNA from an ongoing pregnancy by chorionic villus sampling (CVS) in the first trimester. The spastin gene (SPG4) was completely sequenced. RESULTS: A novel 832insGdelAA frameshift mutation, predicted to cause loss of functional protein, was identified in the affected father and in the fetal DNA. CONCLUSIONS: This is the first report on direct prenatal diagnosis of chromosome 2p-linked AD-HSP (SPG4). In addition, we report a novel SPG4-combined small insertion/deletion mutation in exon 5, which may be the first SPG4 mutational hot spot.     

Prenatal diagnosis of a familial Y long-arm and chromosome 15 short-arm translocation inherited from a mother carrier

ObjectiveWe present prenatal diagnosis of a familial Y long-arm and chromosome 15 short-arm translocation inherited from a mother carrier.Case ReportA 34-year-old primigravid woman underwent amniocentesis at 20 weeks of gestation because of advanced maternal age. Amniocentesis revealed a derived chromosome 15 or 15p+ with an additional material on the short arm of chromosome 15. Cytogenetic analysis of the parents revealed that the phenotypically normal mother carried the same 15p+ variant, and the father had a karyotype of 46,XY. Array comparative genomic hybridization (aCGH) analysis on the DNA extracted from cultured amniocytes revealed no genomic imbalance. Polymorphic DNA marker analysis using the DNAs extracted from cultured amniocytes and parental bloods excluded uniparental disomy (UPD) 15. C-banded preparations and metaphase fluorescence in situ hybridization analysis using a Yq12-specific probe showed a positive stain on the 15p+, indicating the origin of Yq on the short arm of the derivative chromosome 15. The karyotype of amniocentesis was 46,XX,der(15)t(Y;15)(q12;p13)mat. The mother had a karyotype of 46,XX,der(15) t(Y;15)(q12;p13). At 39 weeks of gestation, a 3006-g healthy female baby was delivered with no phenotypic abnormality. During follow-up at age six months, she manifested normal physical and psychomotor development.ConclusionPrenatal diagnosis of a 15p+ variant should include a differential diagnosis of genomic imbalance and UPD 15, and aCGH and polymorphic DNA marker analyses are useful under such a circumstance.     

嵌合体形成及其对产前筛查及诊断结果的影响

随着细胞分子生物学时代的来临,产前筛查和诊断方法也随之发生改变,更多的数据需要正确解读,这对产前诊断医生无疑是一项挑战。其中,染色体嵌合是影响产前筛查与诊断准确性的重要因素之一,受母亲、父亲以及胎盘等多种因素影响,人类普遍存在染色体嵌合,嵌合形成的主要原因是有丝分裂期染色体错误分离。受多种因素的影响,嵌合的临床表现对每个个体都具有唯一性。嵌合对产前筛查和诊断的影响主要为2个方面：①母血中胎儿游离细胞DNA（cell-free fetal DNA,cfDNA）筛查（无创产前筛查）。虽然大量文献报道无创产前筛查对于２１,１８,１３三体有较高的检出率,但由于其检测的母血中cfDNA来源于胎盘,并非真正的胎儿自身DNA,因此当发生胎盘局限性嵌合时会严重影响无创产前筛查结果的准确性。②绒毛染色体核型分析提示存在嵌合时需要行羊膜腔穿刺进行验证。总之,正确认识染色体嵌合的起源、机制和临床结果,可以为病人提供更多有价值的遗传信息,是做好产前筛查与诊断工作并提供遗传咨询的重要前提。     

Non-invasive prenatal diagnosis from the perspective of a low-resource country

Current clinical practice in obstetrics has shifted the paradigm from a conventional prenatal approach based on invasive procedures, risking both fetus and mother, to non-invasive prenatal testing for some fetal conditions via the analysis of cell-free fetal DNA in maternal blood. In the past 15 years, much research has been devoted to refining the methodology for measuring cell-free fetal DNA in maternal circulation and to exploring clinical applications of this technology as a potential tool for prenatal diagnosis. Since the rapid spread around the world of prenatal diagnosis based on cell-free fetal DNA, it is time to start thinking how this cutting-edge technology might influence current practice of obstetrics in low-resource countries.     

Inherited interstitial deletion of chromosomes 5p and 16q without apparent phenotypic effect: further confirmation

We describe two families in which an inherited interstitial deletion is present without apparent associated phenotypic abnormalities. The first deletion was discovered in a 19-year-old male with a previously diagnosed peroxisomal disorder. High-resolution chromosome analysis was interpreted as 46,XY,del(5)(p14.1p14.3). The patient's phenotypically normal mother had the same interstitial deletion. Chromosome 5p14 deletion has been reported in a three-generation family without phenotypic anomalies. We hypothesize that the affected son's phenotype may be coincidental or represent unmasking of an autosomal recessive peroxisomal disorder in the deleted region. The second interstitial deletion was detected by amniocentesis for advanced maternal age. High-resolution chromosome analysis was interpreted as 46,XX,del(16)(q13q22). The same deletion was found in the healthy mother and a normal brother. The pregnancy was carried to term and resulted in the birth of a normal girl. We report these cases as further evidence that rare, unbalanced deletion of specific chromosomal regions may result in no phenotypic effect. Consequences may result from expression of an autosomal recessive disorder on the homologous chromosome. Identification of such deletions is especially important for prenatal diagnosis and genetic counselling.