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Short tandem repeat (STR) analysis to assess chimerism is a critical aspect of routine care particularly in patients facing stem cell transplants but is also relevant in other clinical scenarios. STR analysis provides a means to assess donor and recipient cellular origins in a patient, and, as such, can inform engraftment, rejection, and relapse status in stem cell transplant recipients. In this review of STR testing, the most commonly used method to assess chimerism, its background, procedural details, and clinical utility are discussed. [ABSTRACT FROM AUTHOR]

Accurate diagnosis of primary hyperoxaluria (PH) has important therapeutic consequences. Since biochemical assessment can be unreliable, genetic testing is a crucial diagnostic tool for patients with PH to define the disease type. Patients with PH type 1 (PH1) have a worse prognosis than those with other PH types, despite the same extent of oxalate excretion. The relation between genotype and clinical phenotype in PH1 is extremely heterogeneous with respect to age of first symptoms and development of kidney failure. Some mutations are significantly linked to pyridoxine-sensitivity in PH1, such as homozygosity for p.G170R and p.F152I combined with a common polymorphism. Although patients with these mutations display on average better outcomes, they may also present with CKD stage 5 in infancy. In vitro studies suggest pyridoxine-sensitivity for some other mutations, but confirmatory clinical data are lacking (p.G47R, p.G161R, p.I56N/major allele) or scarce (p.I244T). These studies also suggest that other vitamin B6 derivatives than pyridoxine may be more effective and should be a focus for clinical testing. PH patients displaying the same mutation, even within one family, may have completely different clinical outcomes. This discordance may be caused by environmental or genetic factors that are unrelated to the effect of the causative mutation(s). No relation between genotype and clinical or biochemical phenotypes have been found so far in PH types 2 and 3. This manuscript reviews the current knowledge on the genetic background of the three types of primary hyperoxaluria and its impact on clinical management, including prenatal diagnosis. [ABSTRACT FROM AUTHOR]

Objective: To estimate the incremental yield of single nucleotide polymorphism (SNP) array over karyotype and to assess the diagnostic accuracy of SNP array as a stand-alone test versus SNP array with karyotype in detecting chromosomal abnormalities for prenatal diagnosis in women with an abnormal fetal ultrasound.Study Design: Studies in which SNP array and karyotype had been used for diagnosing chromosomal aberrations in fetuses with abnormal ultrasound findings were included. A systematic search of relevant studies published in the English language in EMBASE, PubMed, CENTRAL, CDSR (Cochrane database of systematic reviews), SCOPUS and Web of science between 1996 and May 2020 was performed. Primary outcome was incremental yield of SNP array over karyotype (number of patients with abnormalities detected by SNP array over number of patients with normal karyotype). Other outcomes included diagnostic accuracy of SNP array alone versus SNP array and karyotype for prenatal diagnosis. Pooled sensitivities, specificities, and their 95% confidence intervals (CI) were presented.Result(s): Seven studies were included. The incremental yield of SNP array over karyotype was 8% (95% Confidence interval, CI 4-12%) while incremental yield of karyotype over SNP array in foetus with abnormal ultrasound was 0% (95% CI 0-1%). The agreement between SNP array and karyotype was 92%. The variant of unknown significance rates ranged between 4 and 8 %. The pooled sensitivity and specificity of SNP array versus SNP array and karyotype was 0.97 (95% CI 0.92-0.99) and 1.00 (95% CI1.00-1.00), respectively.Conclusion(s): SNP array provides additional information on chromosomal abnormalities over and above conventional karyotype. When used as a stand-alone test, SNP array performs favourably as a diagnostic modality when compared against SNP array and karyotype for prenatal diagnosis with an abnormal fetal ultrasound. [ABSTRACT FROM AUTHOR]

Thalassemia is a common genetic disorder. We aimed to present thalassemia mutation data that covers a period of 7 years from the Mediterranean region of Turkey by comparing with hemoglobin indices and to contribute to prenatal diagnosis and genetic counseling studies which should be decided very quickly. In this study, in which a retrospective archive was scanned, the cases were first grouped as α and β thalassemia, and then β thalassemia mutations were examined in a total of 5 groups as UTR-Pro, Codon, IVS, β0, and β+. We have reached the family of the proband that analyzed their Hb indices and genetic mutation. All mutations were statistically compared with Hb indices, HbF, and HbA2. We have identified two new β thalassemia mutations that have the feature of not being defined previously [HBB:C*62 A>G. (3'UTR+1536 A>G) and HBB:C*1 G>A (3'UTR+1475 G>A)]. The most commonly encountered 23 mutations account for 74.7% of all mutations which is unlike the literature. In the β thalassemia group, 73 different mutations were detected. The most common β thalassemia mutation was HBB: c.93-21 G>A (IVS I-110 G>A) with a frequency of 19.72%. A statistically significant difference was found when comparing the mutation groups with Hb indices. We think that it may be useful to evaluate the mutations we have newly identified too together with the Hb indices especially in evaluating the carriers of thalassemia and it will contribute to prenatal diagnosis and genetic counseling studies which should be decided very quickly. [ABSTRACT FROM AUTHOR]

Introduction: The hemoglobinopathies are the commonest group of single gene disorders in the Indian subcontinent. Although genetic modifiers are known to have a remarkable effect on phenotypic expression, the effects of the possible co‐inheritance of different modifiers are not taken into account during prenatal diagnosis. The present study was undertaken to look for the frequency of globin gene modifiers like the types of β‐globin gene mutations, α thalassemia, α gene triplication, and the Xmn1 polymorphism in fetuses during antenatal diagnosis of hemoglobinopathies. Materials and methods: A total of 580 fetuses with different diagnoses were screened for the presence of genetic modifiers. Results: Twenty‐two different β‐globin gene mutations were identified of which 3.5% were milder mutations. Among the affected fetuses, 29.6% of the β‐thalassemia major and 52.9% of the sickle cell anemia (SCA) fetuses had one genetic modifier while 3.7% of the β‐thalassemia major and 41.1% of the SCA fetuses had co‐inherited two modifiers. α‐gene triplication was detected in 16 (3.5%) β‐thalassemia/sickle cell heterozygous and normal fetuses of which 5 babies (2 β‐thalassemia heterozygous and 3 normal) could be followed up. Of the 2 β‐thalassemia heterozygous babies, one had a severe clinical presentation. Conclusion: Many fetuses had one or two gene modifiers. However, the impact of these on ameliorating the severity of the disease could not be evaluated as all the fetuses with β thalassemia major or sickle cell disease were terminated. Parents having heterozygous fetuses with α gene triplication should be followed up periodically after birth for better management of these babies. [ABSTRACT FROM AUTHOR]

Introduction: Invasive prenatal testing with chromosomal microarray analysis may be a relevant option for all pregnant women, but there is only moderate-quality evidence for such an offer. We intended to study the prevalence of copy number variants (CNVs) in prenatal samples using a single SNP-array platform stratified by indication.Material and Methods: A cross-sectional study was performed based on a cohort. From January 2015 to December 2017, a total of 10 377 prenatal samples were received for prenatal single nucleotide polymorphism (SNP)-array in the laboratory of the Genetics Generation Advancement Corporation. Indications for chromosomal microarray analysis studies included the confirmation of an abnormal karyotype, ultrasound abnormalities, advanced maternal age and parental anxiety. CNVs and region of homozygosity identified by the SNP-array were analyzed.Results: Of 10 377 cases, 689 had ultrasound abnormalities and 9688 were ascertained to have other indications. The overall prevalence of CNVs was 2.1% (n = 223/10 377, 95% confidence interval [CI] 1.9-2.4), but the prevalence was 4.4% (95% CI 3.0-6.1) for cases referred with abnormal ultrasound findings and 2.0% (95% CI 1.7-2.3) for other indications. Of the 223 CNVs detected, 42/10 377 were pathogenic (0.4%, 95% CI 0.3-0.6), 84 were susceptibility CNV (0.8%, 95% CI 0.6-1.0) and 97 were variants of uncertain significance (0.9%, 95% CI 0.8-1.1). Using an SNP-based platform allowed for the detection of paternal uniparental disomy of chromosome 14 in a fetus with ultrasound abnormality.Conclusions: With an indication of advanced maternal age but normal ultrasound scans, the prevalence of pathogenic CNVs was 0.4% and that of susceptibility CNV 0.7%. As CNVs are independent of maternal age, the prevalence is likely the same for younger women. Thus, this study provides further evidence that chromosomal microarray analysis should be available for all women who wish to receive diagnostic testing, as this risk is above the cut-off of 1:300 for Down syndrome, leading to the suggestion of invasive testing. A chromosomal microarray analysis based on SNP-array platform is preferable, as it can also detect uniparental disomy in addition to copy number variants. [ABSTRACT FROM AUTHOR]

Due to the high rate of complications, special medical care must be provided especially for monozygotic twin pregnancies, which are characterized as having 2.5 times higher mortality of fetuses. In recent years, examination of cell-free DNA (cfDNA) circulating in maternal plasma has become a useful noninvasive method of prenatal diagnosis. However, fetal DNA constitutes only 3-20% of plasma cfDNA during pregnancy. Short tandem repeats (STRs) are routinely used in forensic examination of DNA mixtures and are able to identify 5% minority components. Haplotypes of deletion/insertion polymorphisms and STRs (DIP-STRs) are able to detect even 0.1% minority components of DNA mixtures. Thus, STRs and DIP-STRs seem to be a perfect tool for detection of fetal alleles in DNA isolated from maternal plasma. Here, we present a novel noninvasive prenatal diagnosis technique of determination of pregnancy zygosity based on examination of feto-maternal microchimerism of plasma cfDNA with the use of STRs and DIP-STRs. Our preliminary results based on 22 STR loci showed 67% sensitivity, 100% specificity and 82% accuracy for prenatal detection of twin dizygosity. The corresponding values for seven DIP-STRs were 13%, 100% and 54%, respectively. Owing to assay performance, low DNA input requirements, low costs (below 10 USD per patient) and simplicity of analysis, genotyping of STR/DIP-STR markers in maternal plasma cfDNA may become a useful supplementary test for noninvasive prenatal diagnosis of twin zygosity in cases when chorionicity and zygosity cannot be reliably determined by ultrasound examination and prognostic value may be provided by a DNA test determining pregnancy zygosity. [ABSTRACT FROM AUTHOR]

Noninvasive prenatal testing (NIPT) utilizes cell-free fetal DNA (cffDNA) present in maternal peripheral blood to detect chromosomal abnormalities. The detection of 21-trisomy, 18-trisomy, and 13-trisomy in the fetus has become a common screening method during pregnancy and has been widely applied in routine clinical testing because of its analytical and clinical validity. Currently, noninvasive prenatal testing involving copy number variations (CNVs) and other frequent single-gene disorders is being widely studied, and it plays an important and indispensable role in prenatal detection. The multiple approaches that have been reported and validated by various laboratories have different merits and limitations. Their clinical validity, utility, and application vary with different diseases. This review summarizes the principles, methods, advantages, and limitations of noninvasive prenatal testing for the detection of aneuploidy, CNVs and single-gene disorders. Before implementation of NIPT into clinical practice, a list of criteria that the application must meet is crucial. Essential parameters such as clinical sensitivity, clinical specificity, positive predictive value (PPV) and negative predictive value (NPV) are required to properly evaluate the clinical validity and utility of NIPT. We then discuss and analyze these clinical parameters and clinical application guidelines, providing physicians and scientists with feasible strategies and the latest research information. [ABSTRACT FROM AUTHOR]

Background: Classical karyotyping and copy-number variation sequencing (CNV-seq) are useful methods for the prenatal detection of chromosomal abnormalities. Here, we examined the potential of using a combination of the two methods for improved and accurate diagnosis. Methods: From February 2013 to January 2018, 64 pregnant women showing indications for fetal chromosomal examination in the affiliated hospital of the Inner Mongolia Medical University were selected for this study. Amniotic fluid was collected and used for karyotype analysis and CNV-seq. Results: Karyotype analysis of the 64 cases showed that six cases (9.38%) had chromosomal abnormalities. Using CNV-seq, in addition to three cases with numerical abnormalities of chromosomes, 14 cases were detected with CNV, of which five were pathogenic CNV, four were of uncertain clinical significance and five were polymorphic CNV. However, CNV-seq failed to detect one case with sex chromosome mosaicism and a balanced translocation carrier. The rate of abnormal chromosome and CNV detection was 26.56% (17/64) by CNV-seq. Conclusion: Application of CNV-seq in prenatal diagnosis could allow the detection of submicroscopic chromosomal abnormalities and effectively reduce the birth of children with microdeletion and microduplication syndrome. Additionally, the combined application of karyotype analysis and CNV-seq can effectively improve the detection rate of chromosome abnormalities. [ABSTRACT FROM AUTHOR]

Introduction: The hemoglobinopathies pose a significant health burden in India. Apart from the β thalassemias and sickle cell disorders, α thalassemias and structural hemoglobin variants are also common. Here we have reviewed the phenotypic and molecular diversity of hemoglobinopathies encountered at a referral center in western India over a period of 15 years. Materials and Methods: Screening for hemoglobinopathies was done using HPLC and cellulose acetate electrophoresis. Molecular characterization was done using Covalent Reverse Dot Blot Hybridization (CRDB), Amplification Refractory Mutation System (ARMS), GAP PCR and direct DNA sequencing. Results: The study includes 31 075 individuals who were referred for diagnosis of hemoglobinopathies and prenatal diagnosis. Of these 14 423 individuals showed various hemoglobin abnormalities. Beta genotyping in 5615 individuals showed the presence of 49 β thalassemia mutations. 143 β thalassemia heterozygotes had normal or borderline HbA2 levels. We identified three δ gene mutations (HbA2 Pellendri, HbA2 St.George, HbA2 Saurashtra) in β thalassemia heterozygotes leading to normal HbA2 levels. The commonest defects among the raised Hb F determinants were Gγ(Αγδβ)0 Indian inversion and the HPFH‐3 Indian deletion. A total of 312 individuals showed the presence of α thalassemia, of which 12.0% had a single α gene deletion (−α/αα). HbH disease was identified in 29 cases with 10 different genotypes. Alpha globin gene triplication was seen in 2.1% of β thalassemia heterozygotes with a thalassemia intermedia phenotype. Seven unusual α chain variants and eight uncommon β chain variants were identified. Conclusion: The repertoire of molecular defects seen in the different globin genes will be valuable for management and control of these disorders both in India as well as in other countries where there is a huge influx of migrant populations from India. [ABSTRACT FROM AUTHOR]

Objective: Noninvasive prenatal diagnosing for fetal trisomy 21 could help to avoid unnecessary amniocentesis. It is feasible to target the cell-free placental-specific mRNA in maternal plasma, thus eliminating the interference from the maternal genetic background.Study Design: In this study, placental-specific genes on chromosome 21 were identified by searching the TiGER database and reviewing the published literature. These cell-free placental-specific mRNA fragments in maternal plasma were amplified and sequenced. Then, the allelic ratio of heterozygous single-nucleotide polymorphisms(SNPs) was analyzed.Results: Four placental-specific genes (DNMT3L, KRTAP26-1, PLAC4, DSCR4) were identified. This study included 42 cases of normal pregnancies and 19 cases of trisomy 21 pregnancies. The probability density function showed that the allelic ratio of heterozygous SNPs in normal pregnancy reached a peak at 1.0, and that in the trisomy 21 pregnancy was a double-peaked curve in both 0.5 and 2.0. However, the allelic ratio of heterozygous SNPs demonstrated no statistical difference between the normal and trisomy 21 pregnancies.Conclusion: Fetal trisomy 21 could not be diagnosed by examining the heterozygous SNPs of cell-free placental-specific mRNA on chromosome 21. This might be because the low quantity and quality of cell-free placental-specific mRNA fragments in maternal plasma, and the limited available heterozygous SNPs on the placental-specific genes. [ABSTRACT FROM AUTHOR]

Background Two common mutations of the solute carrier family 4 member 1 (SLC4A1) gene, namely, Southeast Asian ovalocytosis (SAO) and band 3 Bangkok 1 (G701D), cause autosomal recessive distal renal tubular acidosis (AR dRTA) in ethnic Southeast Asian populations. In this study, we applied the high-resolution melting (HRM) method for screening of AR dRTA associated with SLC4A1 mutations in 10 new patients with unknown cause(s) of AR dRTA. Methods We analyzed SAO and G701D mutations in the patients and their family members using HRM. The results were confirmed by polymerase chain reaction–restriction fragment-length polymorphism (PCR-RFLP) and DNA sequencing techniques. Results All patients carried homozygous G701D mutation, whereas their family members had heterozygous G701D or homozygous wild-type. Conclusions Homozygous G701D is a common cause of AR dRTA in ethnic Thai pediatric populations. HRM can be used as a rapid screening method for common SLC4A1 mutations that cause AR dRTA in Southeast Asian and other populations. [ABSTRACT FROM AUTHOR]

Joubert syndrome (JBTS) is a clinically and genetically heterogeneous group of ciliary diseases. To date, 34 subtypes of JBTS have been classified due to different causative genes or extra clinical features. Most of them are autosomal recessive, while only the subtype 10 (JBTS10) is a quite rare X-linked recessive disorder caused by OFD1 mutations with few reports. In this study, by using whole exome sequencing (WES), a novel OFD1 splicing mutation (c.2488+2T>C) was identified in a male fetus with suspected Dandy-Walker variant (DWV) and syndactyly, for whom abnormal karyotype and pathogenic CNV have been excluded. This mutation was inherited from the mother who has experienced two similar pregnancies before. An abnormal skipping of exon 18 in OFD1 mRNA was confirmed by RT-PCR and sequencing. Result from quantitative RT-PCR also showed that total OFD1 mRNA in the index fetus was significantly lower than the control. After a combined analysis of genetic testing results and genotype-phenotype correlations, the novel mutation c.2488+2T>C in OFD1 was considered to be the genetic cause for the affected fetus. Thus the diagnosis should be JBTS10 rather than the primary clinical diagnosis of DWV. We report the first prenatal case of JBTS10 in Chinese population, which not only helps the family to predict recurrence risks for future pregnancies but also provides more information for understanding such a rare disease. The results also present evidence that WES is an effective method in prenatal diagnosis for those fetuses with Joubert syndrome. [ABSTRACT FROM AUTHOR]

Abstract: Turner syndrome, characterized by the presence of a monosomy X cell line, is a common chromosomal disorder. Patients with Turner syndrome are usually phenotypically female, and male cases are rarely reported. Here, we report a fetus with a mosaic karyotype: mos 45,X/46,X,del(Y)(q11.21). The fetus was initially misdiagnosed as female with Turner syndrome by both noninvasive prenatal testing and cytogenetic analysis of amniotic fluid and was subsequently found to have male anatomy by antenatal ultrasonography at 24 weeks gestational age. Through single nucleotide polymorphism‐array and fluorescence in situ hybridization testing, we found that there was a truncated Y chromosome with sex‐determining region Y (SRY) present in some cells of the fetus, which caused the male features in the fetus. [ABSTRACT FROM AUTHOR]

Purpose: The present study aims to evaluate the utility of high-resolution single-nucleotide polymorphism (SNP) arrays in fetuses with ventricular septal defects (VSDs) with or without other structural anomalies but with normal karyotypes and to investigate the outcomes of cases of prenatal VSDs via clinical follow-up.Methods: We analyzed 144 fetuses with VSDs and normal karyotypes using Affymetrix CytoScan HD arrays and the analyses were carried out a year after birth.Results: Clinically significant CNVs were detected in 12 fetuses (8.3%). The most common pathogenic CNV was a 22q11.2 deletion with a detection rate of 2.8% (4/144). Well-known microdeletion or microduplication syndromes, including Smith-Magenis, Miller-Dieker, 9q subtelomeric deletion, 1p36 microdeletion, 1q21.1 microduplication, and terminal 4q deletion syndrome, were identified in six cases. Three regions of chromosomal imbalance were also identified: microduplication at 12q24.32q24.33, microdeletion at 16p13.13p13.12 and microdeletion at Xp21.1. The genes TBX1, SKI, GJA5, EHMT1, NOTCH1 were identified as established genes and LZTR1, PRDM26, YWHAE, FAT1, AKAP10, ERCC4, and ULK1 were identified as potential candidate genes of fetal VSDs. There was no significant difference in pathogenic CNVs between isolated VSDs and VSDs with additional structural abnormalities. Ninety-five (74.8%) pregnant women with fetuses with benign CNVs chose to continue the pregnancy and had a favorable prognosis, while nine (75%) pregnant women with fetuses with pathogenic CNVs chose to terminate the pregnancy.Conclusions: High-resolution SNP arrays are valuable tools for identifying submicroscopic chromosomal abnormalities in the prenatal diagnosis of VSDs. An excellent outcome can be expected for VSD fetuses that are negative for chromosomal anomalies and other severe anatomic abnormalities. [ABSTRACT FROM AUTHOR]

Objective: Cell-free DNA from maternal plasma can be used for non-invasive prenatal testing for aneuploidies and single gene disorders, and also has applications as a biomarker for monitoring high-risk pregnancies, such as those at risk of pre-eclampsia. On average, the fractional cell-free fetal DNA concentration in plasma is approximately 15%, but can vary from less than 4% to greater than 30%. Although quantification of cell-free fetal DNA is straightforward in the case of a male fetus, there is no universal fetal marker; in a female fetus measurement is more challenging. We have developed a panel of multiplexed insertion/deletion polymorphisms that can measure fetal fraction in all pregnancies in a simple, targeted sequencing reaction. Methods: A multiplex panel of primers was designed for 35 indels plus a ZFX/ZFY amplicon. cfDNA was extracted from plasma from 157 pregnant women, and maternal genomic DNA was extracted for 20 of these samples for panel validation. Sixty-one samples from pregnancies with a male fetus were subjected to whole genome sequencing on the Ion Proton sequencing platform, and fetal fraction derived from Y chromosome counts was compared to fetal fraction measured using the indel panel. A total of 157 cell-free DNA samples were sequenced using the indel panel, and informativity was assessed, along with the proportion of fetal DNA. Results: Using gDNA we optimised the indel panel, removing amplicons giving rise to PCR bias. Good correlation was found between fetal fraction using indels and using whole genome sequencing of the Y chromosome (Spearmans r = 0.69). A median of 12 indels were informative per sample. The indel panel was informative in 157/157 cases (mean fetal fraction 14.4% (±0.58%)). Conclusions: Using our targeted next generation sequencing panel we can readily assess the fetal DNA percentage in male and female pregnancies. [ABSTRACT FROM AUTHOR]

Introduction: Molecular screening of Thalassemia is a burning issue especially for developing country where birth rate is high and most of the victims are from rural and from economically downtrodden part of the society. Methods: The present study reports about the molecular diagnosis and pattern identification of Thalassemia carrier parents and females, who are in certain condition to determine their genotype of Chorionic Villi sample (CVS) to combat birth of new thalassemia baby. Result: Data suggest for the year 2014 and 15 that IVS 1-5(G-C) genotype heterozygous is the most prevalent for each year and affected CVS are either IVS 1-5(G-C) homozygous or compound heterozygous cases containing IVS 1-5(G-C). Bihar, Hooghly, Kolkata, North 24 Parganas, South Dinajpur and North Dinajpur is showing significant difference. Few regions are with a little laxity of patient flow for prenatal diagnosis. Conclusion: This data is useful to make a genetic micro map of West Bengal and also gives the vivid distribution of different Thalassemia gene across the state. [ABSTRACT FROM AUTHOR]

Balanced reciprocal translocations are relatively common human genetic abnormalities that involve the exchange of the terminal segments between different chromosomes and have an approximately 5-80% chance of generating an embryo with chromosomal abnormalities. Non-invasive prenatal testing (NIPT) has been increasingly used in clinical practice to detect fetal trisomies 21, 18 and 13 with a sensitivity and specificity of up to 99%. In this report, we describe a duplication on chromosome 10 and a deletion on chromosome 5 that were first detected on NIPT. Multiple follow-up invasive tests, such as prenatal BACs-on-Beads (BoBs), GTG banding and single nucleotide polymorphism (SNP) array, confirmed the NIPT results. Furthermore, GTG banding identified a normal maternal karyotype and a paternal karyotype with a balanced translocation of 46, XY, t(5;10)(q35.1;p13), inherited by the child. Therefore, NIPT could be a new method for the prenatal diagnosis of fetal chromosomal abnormalities, which, in the present study, were confirmed on multiple clinical and molecular methods as being derived from paternal balanced chromosomal rearrangements. [ABSTRACT FROM AUTHOR]

Objective To assess the additive value of prenatal chromosomal microarray analysis (CMA) for all indications and the likelihood of detecting pathologic copy number variations (CNVs) based on specific indications. Methods A retrospective analysis was performed on amniocentesis and chorionic villi sampling results obtained between 2010 and 2014 in a single institution. A total of 3,314 consecutive patients undergoing invasive genetic testing for different indications were offered CMA in addition to standard karyotype. The prevalence of pathologic CNVs was compared between patients with low-risk indications and those with high-risk indications. Likewise, the prevalence of pathologic CNVs among patients with different sonographic abnormalities was calculated and compared with the low-risk group. Chi-square and Fisher exact tests were used for statistical analysis. Results The prevalence of pathologic CNVs was significantly higher in patients with high-risk indications and specifically those with sonographic abnormalities, compared with the low-risk group (2.8 and 5.9% vs. 0.4%, respectively; all p < 0.05). Conclusion Prenatal CMA detected clinically relevant CNVs in fetuses with a normal karyotype. Major structural malformations and nuchal translucency (NT) ≥ 3.0 mm are associated with the highest risk for a CMA abnormality. Nevertheless, the prevalence of pathologic CNVs in the low-risk population was high enough (1:250) to consider genetic counseling in this group. [ABSTRACT FROM AUTHOR]

Background: This report is about a pregnancy with a triploid fetus and underscores the potential of first trimester combined screening to detect this devastating chromosomal aberration earlier in pregnancy. This report is about a pregnancy with a triploid fetus identified from the first trimester combined screening and confirmed by amniocentesis. Methods: A 28 year old, G5P2AB2 woman was referred to our clinic at 15 weeks of gestation due to a remarkable decrease of her first trimester double biochemical markers and therefore in the high-risk range for trisomy 13 and 18. The woman underwent amniocentesis which revealed a karyotype of 69,XXX. The parents opted for termination and in post mortem physical examination, a hydrocephalus fetus with marked Intra-Uterine Growth Retardation (IUGR) in addition to syndactyly of third and fourth digits, low set malformed ears, micrognathia and club foot, was seen. Results: Our results and previous reports highlight the need to consider a somewhat consistent pattern of the first trimester combined screening in a pregnancy with triploidy and underscore the potential of this screening strategy to detect this chromosomal aberration earlier in pregnancy. Conclusion: Early prenatal diagnosis of this syndrome would provide women an opportunity to terminate an affected pregnancy earlier. This is also important in preventing the risks of associated later induced abortion or obstetric complications. [ABSTRACT FROM AUTHOR]

Objective: To validate an updated version (Version 2) of a single-nucleotide polymorphism (SNP)-based noninvasive prenatal test (NIPT) and to determine the likelihood of success when testing for fetal aneuploidies following a redraw.Methods: Version 2 was analytically validated using 587 plasma samples with known genotype (184 trisomy 21, 37 trisomy 18, 15 trisomy 13, 9 monosomy X, 4 triploidy and 338 euploid). Sensitivity, specificity and no-call rate were calculated, and a fetal-fraction adjustment was applied to enable projection of these values in a commercial distribution. Likelihood of success of a second blood draw was computed based on fetal fraction and maternal weight from the first draw.Results: Validation of this methodology yielded high sensitivities (≥99.4%) and specificities (100%) for all conditions tested with an observed no-call rate of 2.3%. The no-call threshold for sample calling was reduced to 2.8% fetal fraction. The redraw success rate was driven by higher initial fetal fractions and lower maternal weights, with the fetal fraction being the more significant variable.Conclusions: The enhanced version of this SNP-based NIPT method showed a reduced no-call rate and a reduced fetal-fraction threshold for sample calling in comparison to the earlier version, while maintaining high sensitivity and specificity. [ABSTRACT FROM AUTHOR]

Purpose: The aim of this study was to investigate the relationships between the maternal levels of oxidized LDL (ox-LDL), lipid peroxidation marker malondialdehyde (MDA) and LOX-1 3'UTR188C/T and K167N single nucleotide polymorphisms in pregnant Turkish women with gestational diabetes mellitus (GDM).Methods: 116 pregnant women with GDM and 120 healthy pregnant women from the same geographic region were included in the study. Polymerase chain reaction-based restriction analysis was used to identify 3'UTR188C/T and K167N polymorphisms of the LOX-1 gene. Plasma ox-LDL and MDA levels were determined by enzyme-linked immunosorbent assay and spectrophotometric method in all study subjects, respectively.Results: Our results indicated that the distribution of the LOX-1 3'UTR188C/T and K167N genotypes and alleles did not differ significantly among subjects with or without GDM (p > 0.05). TT and NN genotype carriers are associated with some glucose metabolism parameters (p < 0.05). There were no significant differences among plasma ox-LDL and MDA levels with regard to LOX-1 3'UTR188C/T and K167N polymorphisms in GDM group and control subjects (p > 0.05). According to the combined genotype analysis of LOX-1 3'UTR 188 TT and K167N NN polymorphisms, plasma MDA and ox-LDL levels were significantly different between women with GDM and healthy subjects either with or without combined TT/NN genotype carriers (p < 0.001).Conclusions: According to our results, ox-LDL and MDA levels were increased in GDM pregnant women and healthy pregnant women either with or without combined TT/NN genotype carriers, for our Turkish sample, these genotype carriers appear to be related with increased oxidative stress in patients with GDM. [ABSTRACT FROM AUTHOR]

Objectives: To evaluate the performance of a single-nucleotide polymorphism (SNP)-based non-invasive prenatal test (NIPT) for the detection of fetal 22q11.2 deletion syndrome in clinical practice, assess clinical follow-up and review patient choices for women with high-risk results.Methods: In this study, 21 948 samples were submitted for screening for 22q11.2 deletion syndrome using a SNP-based NIPT and subsequently evaluated. Follow-up was conducted for all cases with a high-risk result.Results: Ninety-five cases were reported as high risk for fetal 22q11.2 deletion. Diagnostic testing results were available for 61 (64.2%) cases, which confirmed 11 (18.0%) true positives and identified 50 (82.0%) false positives, resulting in a positive predictive value (PPV) of 18.0%. Information regarding invasive testing was available for 84 (88.4%) high-risk cases: 57.1% (48/84) had invasive testing and 42.9% (36/84) did not. Ultrasound anomalies were present in 81.8% of true-positive and 18.0% of false-positive cases. Two additional cases were high risk for a maternal 22q11.2 deletion; one was confirmed by diagnostic testing and one had a positive family history. There were three pregnancy terminations related to screening results of 22q11.2 deletion, two of which were confirmed as true positive by invasive testing.Conclusions: Clinical experience with this SNP-based non-invasive screening test for 22q11.2 deletion syndrome indicates that these deletions have a frequency of approximately 1 in 1000 in the referral population with most identifiable through this test. Use of this screening method requires the availability of counseling and other management resources for high-risk pregnancies. [ABSTRACT FROM AUTHOR]

Objectives: To evaluate the usefulness of single-nucleotide polymorphism (SNP) array for prenatal genetic diagnosis of congenital heart defect (CHD), we used this approach to detect clinically significant copy number variants (CNVs) in fetuses with CHDs.Methods: A HumanCytoSNP-12 array was used to detect genomic samples obtained from 39 fetuses that exhibited cardiovascular abnormalities on ultrasound and had a normal karyotype. The relationship between CNVs and CHDs was identified by using genotype-phenotype comparisons and searching of chromosomal databases. All clinically significant CNVs were confirmed by real-time PCR.Results: CNVs were detected in 38/39 (97.4%) fetuses: variants of unknown significance were detected in 2/39 (5.1%), and clinically significant CNVs were identified in 7/39 (17.9%). In 3 of the 7 fetuses with clinically significant CNVs, 3 rare and previously undescribed CNVs were detected, and these CNVs encompassed the CHD candidate genes FLNA (Xq28 dup), BCOR (Xp11.4 dup), and RBL2 (16q12.2 del).Conclusion: Compared with conventional cytogenetic genomics, SNP array analysis provides significantly improved detection of submicroscopic genomic aberrations in pregnancies with CHDs. Based on these results, we propose that genomic SNP array is an effective method which could be used in the prenatal diagnostic test to assist genetic counseling for pregnancies with CHDs. [ABSTRACT FROM AUTHOR]

Objectives: To compare the performance of traditional G-banding karyotyping with that of copy number variation sequencing (CNV-Seq) for detection of chromosomal abnormalities associated with miscarriage.Methods: Products of conception (POC) were collected from spontaneous miscarriages. Chromosomal abnormalities were detected using high-resolution G-banding karyotyping and CNV sequencing. Quantitative fluorescent polymerase chain reaction analysis of maternal and POC DNA for short tandem repeat (STR) markers was used to both monitor maternal cell contamination and confirm the chromosomal status and sex of the miscarriage tissue.Results: A total of 64 samples of POC, comprising 16 with an abnormal and 48 with a normal karyotype, were selected and coded for analysis by CNV-Seq. CNV-Seq results were concordant for 14 (87.5%) of the 16 gross chromosomal abnormalities identified by karyotyping, including 11 autosomal trisomies and three sex chromosomal aneuploidies (45,X). Of the two discordant results, a 69,XXX polyploidy was missed by CNV-Seq, although supporting STR marker analysis confirmed the triploidy. In contrast, CNV-Seq identified a sample with 45,X karyotype as a 45,X/46,XY mosaic. In the remaining 48 samples of POC with a normal karyotype, CNV-Seq detected a 2.58-Mb 22q deletion associated with DiGeorge syndrome and nine different smaller CNVs of no apparent clinical significance.Conclusions: CNV-Seq used in parallel with STR profiling is a reliable and accurate alternative to karyotyping for identifying chromosome copy number abnormalities associated with spontaneous miscarriage. [ABSTRACT FROM AUTHOR]

Background: Noninvasive prenatal testing can be used to accurately detect chromosomal aneuploidies in circulating fetal DNA; however, the necessity of parental haplotype construction is a primary drawback to noninvasive prenatal diagnosis (NIPD) of monogenic disease. Family-specific haplotype assembly is essential for accurate diagnosis of minuscule amounts of circulating cell-free fetal DNA; however, current haplotyping techniques are too time-consuming and laborious to be carried out within the limited time constraints of prenatal testing, hampering practical application of NIPD in the clinic. Here, we have addressed this pitfall and devised a universal strategy for rapid NIPD of a prevalent mutation in the Ashkenazi Jewish (AJ) population.Methods: Pregnant AJ couples, carrying mutation(s) in GBA, which encodes acid β-glucosidase, were recruited at the SZMC Gaucher Clinic. Targeted next-generation sequencing of GBA-flanking SNPs was performed on peripheral blood samples from each couple, relevant mutation carrier family members, and unrelated individuals who are homozygotes for an AJ founder mutation. Allele-specific haplotypes were constructed based on linkage, and a consensus Gaucher disease-associated founder mutation-flanking haplotype was fine mapped. Together, these haplotypes were used for NIPD. All test results were validated by conventional prenatal or postnatal diagnostic methods.Results: Ten parental alleles in eight unrelated fetuses were diagnosed successfully based on the noninvasive method developed in this study. The consensus mutation-flanking haplotype aided diagnosis for 6 of 9 founder mutation alleles.Conclusions: The founder NIPD method developed and described here is rapid, economical, and readily adaptable for prenatal testing of prevalent autosomal recessive disease-causing mutations in an assortment of worldwide populations.Funding: SZMC, Protalix Biotherapeutics Inc., and Centogene AG. [ABSTRACT FROM AUTHOR]

A genetic etiology for autism spectrum disorders (ASDs) was first suggested from twin studies reported in the 1970s. The identification of gene mutations in syndromic ASDs provided evidence to support a genetic cause of ASDs. More recently, genome-wide copy number variant and sequence analyses have uncovered a list of rare and highly penetrant copy number variants (CNVs) or single nucleotide variants (SNVs) associated with ASDs, which has strengthened the claim of a genetic etiology for ASDs. Findings from research studies in the genetics of ASD now support an important role for molecular diagnostics in the clinical genetics evaluation of ASDs. Various molecular diagnostic assays including single gene tests, targeted multiple gene panels and copy number analysis should all be considered in the clinical genetics evaluation of ASDs. Whole exome sequencing could also be considered in selected clinical cases. However, the challenge that remains is to determine the causal role of genetic variants identified through molecular testing. Variable expressivity, pleiotropic effects and incomplete penetrance associated with CNVs and SNVs also present significant challenges for genetic counseling and prenatal diagnosis. [ABSTRACT FROM AUTHOR]

Our study aimed to determine the number of couples with normal hemoglobin (Hb) electrophoresis and low-borderline hematological values, which may come up with a clinically critical status in their offspring. The number of couples at risk for severe α-thalassemia (α-thal) needed to be estimated before recommending genetic counseling and prenatal diagnosis (PND). During the past 14 years, from at least 7000 referrals, 754 couples were investigated for α-thal by direct mutation detection methods followed by reverse strip assay and α-globin gene sequencing for inconclusive cases. Detection of silent β-thalassemia (β-thal) mutations was done in suspected cases by complete β-globin gene sequencing. We were able to provide a molecular diagnosis in 87.3% (658/754) of couples. A total of 9.1% (60/658) may have a clinically significant hemoglobinopathy in their offspring. Significant conditions included hydrops fetalis (20.0%; 12/60), certain Hb H (β4) genotypes (78.3%; 47/60) and β-thal intermedia (β-TI) (1.7%; 1/60). The diagnostic flowchart for couples with microcytic hypochromic anemia in countries with a high prevalence of hemoglobinopathies should include α and β gene sequencing. As our results indicate, every nine out of 100 of these couples will face significant hemoglobinopathies and every two out of 100 can carry Hb Bart's (γ4) hydrops fetalis in their pregnancies. For such cases, PND should be utilized to allow the carrier couples to decide whether or not to abort the fetus. [ABSTRACT FROM AUTHOR]

ABSTRACT Non-invasive prenatal testing ( NIPT) for aneuploidy using cell-free DNA in maternal plasma is revolutionizing prenatal screening and diagnosis. We review NIPT in the context of established screening and invasive technologies, the range of cytogenetic abnormalities detectable, cost, counseling and ethical issues. Current NIPT approaches involve whole-genome sequencing, targeted sequencing and assessment of single nucleotide polymorphism ( SNP) differences between mother and fetus. Clinical trials have demonstrated the efficacy of NIPT for Down and Edwards syndromes, and possibly Patau syndrome, in high-risk women. Universal NIPT is not cost-effective, but using NIPT contingently in women found at moderate or high risk by conventional screening is cost-effective. Positive NIPT results must be confirmed using invasive techniques. Established screening, fetal ultrasound and invasive procedures with microarray testing allow the detection of a broad range of additional abnormalities not yet detectable by NIPT. NIPT approaches that take advantage of SNP information potentially allow the identification of parent of origin for imbalances, triploidy, uniparental disomy and consanguinity, and separate evaluation of dizygotic twins. Fetal fraction enrichment, improved sequencing and selected analysis of the most informative sequences should result in tests for additional chromosomal abnormalities. Providing adequate prenatal counseling poses a substantial challenge given the broad range of prenatal testing options now available. Copyright © 2013 ISUOG. Published by John Wiley & Sons Ltd. [ABSTRACT FROM AUTHOR]

Abstract: Endothelial NO, which is synthesized by endothelial nitric oxide synthase (eNOS), has been reported to be related with the occurrence of pre-eclampsia (PE). However, the polymorphisms of eNOS (−786T>C, 4b/a and G894T), the level of nitric oxide and the risk of PE remain unclear. Thus we performed this meta-analysis to determine the associations between them in order to predict the risk for PE and interference with PE development in the early period of antenatal care. All studies investigating the associations between PE risk and polymorphisms of eNOS, or PE risk and serum concentration of NO were reviewed. Finally, 29 studies were included, involving 11 for −786T>C, 11for 4b/a, and 22 for G894T polymorphisms and PE risk. In the overall analysis, −786T>C polymorphism was found to be related with increased PE risk in the dominant model (OR=1.17, 95% CI=1.02-1.35). a allele for 4b/a suffers the high risk of PE (OR=1.46, 95% CI=1.01–2.10). In the subgroup analysis, significantly increased risk was detected among Europeans for −786T>C polymorphism (OR=1.40, 95%CI=1.14–1.73).However, no significant association was detected for G894T polymorphism in the overall and subgroup analysis. The comprehensive evaluation of 9 available studies indicated that serum NO level was significantly decreased in case group (SMD=−0.96umol/mL, 95%CI=−1.80, −0.12umol/mL).Hence, we concluded that eNOS gene −786T>C and 4b/a except for G894T polymorphisms were contributed significantly to PE risk, especially for Europeans, and a low NO concentration in serum increased the risk for PE. [Copyright &y& Elsevier]

Background: Hemoglobinopathy and thalassemia are prevalent genetic disorders throughout the world. Beta thalassemia is one of these disorders with high prevalence in Iran, especially in Khuzestan province. In this study, the rate of different mutations in β-globin gene for prenatal diagnosis in fetal samples was evaluated. Materials and methods: In this experimental pilot study, 316 fetal samples (chorionic villus or amniotic fluid) suspicious to hemoglobin disorders were enrolled. Afterwards, DNA was extracted and PCR and DNA sequencing were used for evaluation of different mutations in β-globin gene. Results: Amongst 316 samples evaluated for prenatal diagnosis, 180 cases (56.8%) were carrying at least one mutated gene of β-thalassemia. In addition, results showed that CD 36-37 (- T) and IVS II-1 (G>A) polymorphisms are the most prevalent polymorphisms of β-thalassemia in Ahvaz city with 13.9% and 10.1% rates, respectively. Conclusion: Using molecular tests for prenatal diagnosis is considered an efficient approach for reducing the birth of children with hemoglobinopathy and identification of prevalent mutations in each region. [ABSTRACT FROM AUTHOR]

Background: Cystic fibrosis (CF) is a multiorganic autosomal recessive disorder, caused by mutation in cystic fibrosis transmembrane conductance regulator (CFTR). CF is highly heterogeneous in Iranian population and molecular diagnosis based on direct identification of mutations is not completely efficient. The use of polymorphic intragenic markers not only can facilitate phenotype prediction in prenatal diagnosis by gene tracking, but also can lead to the demonstration of possible associations between haplotypes and specific mutations. Methods: 60 CF patients and 53 fertile normal subjects originating from North of Iran were analyzed for F508del mutation and c.1210-12T(5_9), c.1408A>G and c.744-33GATT(6_8) polymorphisms. Results: c.1210-12T[7] is the most prevalent allele in normal individuals and CF non-F508del patients with 87.7%and 86.7% frequencies respectively. c.1408A>G survey showed that frequency of allele G and A is nearly equal in both non-F508del CF patients and normal individuals. c.744-33GATT(6_8) study showed that 7 repeat is the most prevalent allele in normal individuals and non-F508del CF patients with 80.2% and 82.1% frequencies respectively. The [c.1408A; c.1210-12T[9]; c.744-33GATT[6]] haplotype was only associated with mutant alleles including F508del. Conclusions: The allelic distribution and heterozygosity results suggest that c.1408A>G, c.1210-12T(5_9) and c.744-33GATT(6_8) can contribute to carrier detection and prenatal diagnosis of CF in Iranian families with previous history of the disease. [ABSTRACT FROM AUTHOR]

Abstract: The short tandem repeats (STR) are undoubtedly the most used molecular markers in genetic diversity studies. β-thalassemia is a hereditary hemolytic anemia which is a common health problem all over the world. The disease has two main clinical forms: the severe form or β-thalassemia major (TM) and the moderate form or β-thalassemia intermedia (TI). To help understand this inconsistency, five STR have been studied in a group of 27 β-thalassemic patients divided into 15 (TM) and 12 (TI). For each STR, the distribution of alleles, has been compared among patients with β-thalassemia major (TM) and those with β-thalassemia intermedia (TI).The comparison has shown, for the STR D14S72, one specific allele to (TM) and for the STR D14S990 and D14S68, two specific alleles to TI patients. The combination of these alleles with severe beta-thalassemia genotypes leads to the phenotype of beta-thalassemia intermedia even though there are no any attenuating factors such as XmnI Gg polymorphism at position −158 of the HBG2 promotor or the alpha-globin defects. This suggests that these alleles are associated with factors reducing β-thalassemia clinical effects. One of this STR is located in the 5′UTR of the CEBPε gene which incodes a transcription factor expressed in the myeloid lineage. Thus, the involvement of the transcription factor CEBPε in the myeloid cells suggests that CEBPε is a candidate gene involved in the modulation of the β-thalassemia expression through immune system reactions. [Copyright &y& Elsevier]

Objective: Four combinations of five neutral sequence changes at rs713040, rs10768683, rs7480526, rs7946748, and rs1609812 occurring in the human beta globin gene defined as frameworks have been reported in beta globin gene. Here we report for the frequency of these frameworks in thalassemia major patients of North Iran. Methods: Beta globin gene frameworks of 46 thalassemia major patients of North Iran were determined using Denaturing Gradient Gel Electrophoresis. Findings: All these frameworks called framework 1, 2, 3, 3a were present at the frequency of 23.9%, 45.7%, 6.5% and 23.9% respectively. Conclusion: These frameworks may be used for tracking mutant alleles in prenatal diagnosis programs. [ABSTRACT FROM AUTHOR]

Abstract: Background: X-linked Adrenoleukodystrophy (X-ALD), with an incidence of 1:14,000 is the most frequent monogenic demyelinating disorder worldwide. The principal biochemical abnormality in X-ALD is the increased levels of saturated, unbranched very long chain fatty acids (VLCFA). It is caused by mutations in ABCD1 gene. No molecular data on X-ALD is available in India and mutational spectrum in Indian patients is not known. Methods: We standardized conformation sensitive gel electrophoresis (CSGE) method to detect mutations in ABCD1 gene in twenty Indian patients with X-ALD. The results were confirmed by sequencing. Genotype–phenotype correlation was also attempted. Prenatal diagnosis (PND) in one family was done using chorionic villi (CV) sample at 12weeks of gestation. Results: Out of twenty, causative mutations could be identified in twelve patients (60%). Six reported and four novel mutations were identified. Three polymorphisms were also observed. No hot spot was found. No significant genotype–phenotype correlation could be established. Conclusions: The study identified the mutation spectrum of Indian X-ALD patients, which enabled us to offer accurate genetic counseling, carrier detection and prenatal diagnosis where needed. [Copyright &y& Elsevier]

Duchenne and Becker muscular dystrophies (DMD/BMD) are allelic, x-linked neuromuscular disease resulting from mutations in dystrophin gene. DMD is the most severe and frequent inherited but still incurable disease in males. About two-third of such patients have large deletions or duplications that can be identified by multiplex polymerase chain reaction (PCR). In one-third of remaining cases, a linkage analysis that involves DNA markers of intragenic dystrophic gene is considered a rapid and simple method for carrier detection and prenatal diagnosis. In the present study, we investigated frequency and heterozygosity of three polymorphic restriction sites and also four highly polymorphic (CA) n repeat microsatellites loci within hot spots region of human dystrophin gene in 60 healthy Iranian populations. Our findings indicated that the allele frequencies of pERT87-8/TaqI, pERT87-15/BamHI, and pERT87-15/XmnI were 0.23/0.77, 0.221/0.779, and 0.239/0.761, respectively. Among these three polymorphic sites, pERT78-15/XmnI locus had the highest heterozygosity with frequency of 47.17%%. We also found that STR49 had the highest heterozygosity among four polymorphic microsatellites. These findings are useful in linkage analysis of Iranian DMD families in both carrier detection and prenatal diagnosis. [ABSTRACT FROM AUTHOR]

Reliable detection of large deletions from cell-free fetal DNA (cffDNA) in maternal plasma is challenging, especially when both parents have the same deletion owing to a lack of specific markers for fetal genotyping. In order to evaluate the efficacy of a non-invasive prenatal diagnosis (NIPD) test to exclude a-thalassemia major that uses SNPs linked to the normal paternal a-globin allele, we established a novel protocol to reliably detect paternal SNPs within the (--SEA) breakpoints and performed evaluation of the diagnostic potential of the protocol in a total of 67 pregnancies, in whom plasma samples were collected prior to invasive obstetrics procedures in southern China. A group of nine SNPs identified within the deletion breakpoints were scanned to select the informative SNPs in each of the 67 couples DNA by multiplex PCR based minisequencing technique. The paternally inherited SNP allele from cffDNA was detected by allele specific real-time PCR. A protocol for reliable detection of paternal SNPs within the (--SEA) breakpoints was established and evaluation of the diagnostic potential of the protocol was performed in a total of 67 pregnancies. In 97% of the couples one or more different SNPs within the deletion breakpoint occurred between paternal and maternal alleles. Homozygosity for the (--SEA) deletion was accurately excluded in 33 out of 67 (49.3%, 95% CI, 25.4-78.6%) pregnancies through the implementation of the protocol. Protocol was completely concordant with the traditional reference methods, except for two cases that exhibited uncertain results due to sample hemolysis. This method could be used as a routine NIPD test to exclude gross fetal deletions in a-thalassemia major, and could further be employed to test for other diseases due to gene deletion. [ABSTRACT FROM AUTHOR]

BACKGROUND Cell-free fetal DNA (cff DNA) and RNA can be detected in maternal plasma and used for non-invasive prenatal diagnostics. Recent technical advances have led to a drastic change in the clinical applicability and potential uses of free fetal DNA and RNA. This review summarizes the latest clinical developments in non-invasive prenatal diagnosis in the context of the latest technical developments. METHODS We searched PubMed with the search terms ‘prenatal’, ‘non-invasive’, ‘fetal DNA’, ‘mRNA’ and cross-referenced them with ‘diagnostics’, ‘microRNA’, ‘aneuploidy’, ‘trisomy’ and ‘placenta’. We also searched the reference list of the articles identified by this search strategy. RESULTS Genome-wide methods have been, or can be, successfully applied on total DNA (DNA-seq), methylated DNA immunoprecipitation (with tiling array), microRNA (Megaplex) and total RNA (RNA-seq). Chromosome- or gene-specific assays have been successively applied on placenta RNA (allele ratio) or DNA multiplex ligation-dependent probe amplification (MLPA). These methods are reviewed for their merits and pitfalls with consideration of the placental biology. For the purpose of clarity, the technical and clinical characteristics are limited to non-invasive prenatal detection of chromosomal aneuploidies, with emphasis on trisomy 21. CONCLUSIONS The technical advances for non-invasive aneuploidy tests based on cff DNA and placental mRNA in maternal plasma have been enormous. Multimarker assays including genome-wide approaches with the option of qualitative information on variation (polymorphism or mutation) besides quantitative information are the preferred methods of choice. The time for population-based, double blind, large-scale clinical cohort trials has come. [ABSTRACT FROM AUTHOR]

Mucopolysaccharidosis type IVA or Morquio A syndrome is characterized by the lack of N-acetylgalactosamine-6-sulfate-sulfatase and the accumulation of keratan sulfate and chondroitin-6-sulfate in the lysosomes. At least, 148 mutations and 16 polymorphisms were identified in the GALNS gene. The aim of this study was the screening of polymorphisms within 7 patients recruited from many regions of Tunisia in order to determine the haplotypes and their association with the mutations previously reported. Patients and methods: We have used the PCR sequencing to analyse the different haplotypes and to identify the polymorphisms within 7 affected MPS IVA patients. Results: Nine GALNS polymorphisms were detected in the 7 studied patients. Five of these polymorphisms are within the GALNS gene exons. Six polymorphisms have been previously described and used for linkage analysis in MPS IVA patients and determination of haplotypes. We have identified two novel heterozygous polymorphisms in intron 13 and intron 3 Conclusion: Polymorphisms may be useful for carrier detection and prenatal diagnosis in informative families whose specific mutations have not been identified. The determination of haplotypes can also determine the origin of some mutations in a population. [ABSTRACT FROM AUTHOR]

Abstract: We describe the outcome of two consecutive pregnancies with a clinical presentation of ornithine transcarbamylase (OTC) deficiency (OTCD) without a molecular diagnosis. A 119kb deletion on Xp11.4 including the OTC gene was detected in the mother. The same deletion was identified in the blood spots from deceased male newborns. In patients with a clinical and biochemical presentation of OTCD and negative OTC sequencing, whole genome or targeted chromosomal microarray analysis (CMA) with coverage of the OTC and neighboring genes should be performed as a reflex test. [ABSTRACT FROM AUTHOR]

Abstract: Objective: To evaluate in maternal plasma, the efficacy of detecting the paternal β-gene mutation and informative single nucleotide polymorphisms (SNPs) linked to the paternal-mutant or -normal allele in non-invasive prenatal diagnosis (NIPND). Study design: In 20 at-risk pregnancies, using the allele-specific arrayed primer extension (AS-APEX) technology of the previously published “Thalassemia” array, cyanine-5-deoxycytosine triphosphate (Cy5-dCTP) was incorporated into the extended strands to matched PCR-amplified maternal plasma DNA templates, to detect both the paternal β-gene mutation and informative paternal SNPs. Results: Sensitivity experiment showed that 5pg DNA as starting template gave detectable signals on the array. In 13 cases (65%), the paternal-derived β-gene mutation and/or informative mutant-associated SNP were detected. A subsequent invasive procedure was required to determine if the fetus had a β-thalassemia (thal) major or minor genotype. In 3 cases (15%), absence of the paternal mutant or mutant-associated SNP excluded a β-thal major fetus; while in 4 cases (20%), this approach was non-discriminative as both parents carry the same mutation without any informative SNP. Conclusion: This approach was useful in 16 out of 20 (80%) pregnancies at risk for β-thal in southern Chinese. [Copyright &y& Elsevier]

β-Thalassemia (β-thal) is a major health problem in Iran and the incidence of carriers is around 3–4%. The disease is caused by heterogeneous mutations in the β-globin gene and is characterized by hypochromic microcytic anemia. The human β-globin complex spans a region of 70 kb and contains over 20 restriction fragment length polymorphisms (RFLPs). At least nine RFLP markers including RsaI/β in the β-globin gene cluster have been routinely exploited for prenatal diagnosis. Here, we report a novel polymorphism upstream of the β-globin gene characterized by RsaI digestion. Sequencing of a fragment containing this area showed a nucleotide change (T>C) at position −223 upstream of the β-globin gene. This change could interfere with precise interpretation of the RsaI digestion pattern in linkage analysis and prenatal diagnosis of β-thal. [ABSTRACT FROM AUTHOR]

Background: A high number of mutations associated with cystic fibrosis have been identified in the cystic fibrosis transmembrane conductance regulator ( CFTR) gene, most of which are rare and, therefore, hamper extensive molecular diagnosis. In couples undergoing prenatal diagnosis where no mutation is found in one or both partners, additional analysis of intragenic polymorphisms may allow for the identification of fetal alleles associated with cystic fibrosis. Methods: We developed novel, rapid and accurate assays for CFTR genotype determination using pyrosequencing technology; a simple, automated and reliable technique with low cost. Results: Assays were optimized for the identification of the seven most frequent CFTR mutations ( p.DeltaF508, p.N1303K, p.G542X, c.2183AA>G, c.1717-1G>A, p.W1282X, p.R1162X) in the Italian population and two common intragenic polymorphisms ( rs213950 and rs1800136). Blind validation on 15 known control samples, typed for each sequence variation, allowed correct identification of all 135 genotypes. Conclusions: We demonstrated that this procedure is highly specific for the identification of individual CFTR sequence variations associated with cystic fibrosis, allowing both population screening and prenatal diagnosis. Clin Chem Lab Med 2009;47:1051–4. [ABSTRACT FROM AUTHOR]

Background: The identification of very low-levels of minority sequences has interesting clinical and diagnostic applications. Among these, non-invasive prenatal diagnosis of genetic diseases on fetal DNA circulating in maternal plasma is an emerging field of application. Methods: A combined approach based on innovative microarray slides coated with a special copolymer was developed for the identification of three polymorphisms located in the causative gene for cystic fibrosis (CF). This technique was applied to the analysis of fetal DNA in maternal plasma from four couples that carried different allelic variants. Results: The use of highly sensitive slides correctly identified fetal paternally inherited alleles without any enrichment strategy. Conclusions: Our results confirm that the high fluorescence signal provided by the optimized substrate may be applied to the identification of any fetal paternally inherited sequence. This helps extend the application of non-invasive prenatal diagnosis to genetic diseases caused by predominant mutations or minor rare molecular defects. Clin Chem Lab Med 2009;47:818–23. [ABSTRACT FROM AUTHOR]

The aim of this pilot study was to assess the association between polymorphisms in genes that encode for proteins involved in the pharmacokinetics/pharmacodynamics of glucocorticoids and the occurrence of respiratory distress syndrome (RDS) in preterm infants born to mothers treated with a complete course of betamethasone. Sixty-two preterm infants were enrolled. The C1236T, G2677T, and C3435T polymorphisms in the ABCB1 gene, BclI, N363S and ER22/23EK in the NR3C1 gene, I105V in the GST-P1 gene and GST-M1 and GST-T1 deletions were analyzed, and their association with the occurrence of RDS was assessed. In univariate analysis, the heterozygous and homozygous presence of the I105V variant in the GST-P1 gene seemed to confer protection against the occurrence of RDS ( P = 0.032), while no association for all other polymorphisms was observed. In multivariate analysis, selection from the reference model of independent variables based on AIC (Akaike information criteria) maintained three variables in the model: gestation, C3435T, and GST-P1 genotype. Polymorphisms of the GST-P1 gene may influence the effect of antenatal steroids on the newborn lung. [ABSTRACT FROM AUTHOR]

The integrin GPIIb-IIIa plays an important role in platelet activation and plug formation. Quantitative and qualitative dysfunction in this receptor causes Glanzmann's thrombasthenia (GT) and leads to a bleeding tendency. Mutations in the GPIIb or GPIIIa gene are known to be responsible for the inherited form of this disease, which is characterized by mucocutaneous bleeding and other clinically severe bleeding manifestations. GT is an autosomal inherited platelet function. Mutations in the GPIIIa gene but not GPIIb gene in GT patients from Western India have been studied. Hence, this study was designed and included for the first time two mutation detection strategies: single strand conformation polymorphism and conformation sensitive gel electrophoresis followed by sequencing. Patients diagnosed as GT in our laboratory were screened for mutations. We report 12 mutations in 13 patients, of which 11 are novel mutations. Six mutations were missense, four were deletions and two were splice junction mutations. The study thus identifies novel mutations in the GPIIb gene in patients from Western India, implicating the importance of certain amino acids in structure-function correlations as well as enabling prenatal diagnosis in these families. [ABSTRACT FROM AUTHOR]

Abstract: Objective: The objective was to evaluate the beta-globin gene mutations and polymorphisms in cell-free fetal DNA in the early first trimester (7–9th weeks’ gestation) for the prediction of thalassemia risk at chorionic villous sampling (CVS). Study design: Beta-globin gene mutations and polymorphisms were analyzed in 97 carrier families and 100 control couples. Using conventional PCR-DGGE we carried out cell-free fetal DNA analysis in 37 couples in whom only the father was an IVSI-110 carrier. Results: Beta-globin gene mutations have 80% information content in contrast to 39% of polymorphisms. By non-invasive early first-trimester identification of the paternally transmitted IVSI-110 mutation, we reached a sensitivity and specificity of 96 and 100%, respectively. Although the detection rate of the Y chromosome in male fetuses was as high as 100%, beta-thalassemia allele drop-out cannot be excluded. Conclusions: Even though there is high sensitivity in non-invasive paternally transmitted beta-thalassemia mutation detection, intense effort must be made to avoid misdiagnoses before the clinical application of this approach. [Copyright &y& Elsevier]

The discovery of fetal mRNA transcripts in the maternal circulation holds great promise for noninvasive prenatal diagnosis. To identify potential fetal biomarkers, we studied whole blood and plasma gene transcripts that were common to 9 term pregnant women and their newborns but absent or reduced in the mothers postpartum. RNA was isolated from peripheral or umbilical blood and hybridized to gene expression arrays. Gene expression, paired Student's t test, and pathway analyses were performed. In whole blood, 157 gene transcripts met statistical significance. These fetal biomarkers included 27 developmental genes, 5 sensory perception genes, and 22 genes involved in neonatal physiology. Transcripts were predominantly expressed or restricted to the fetus, the embryo, or the neonate. Real-time RT-PCR amplification confirmed the presence of specific gene transcripts; SNP analysis demonstrated the presence of 3 fetal transcripts in maternal antepartum blood. Comparison of whole blood and plasma samples from the same pregnant woman suggested that placental genes are more easily detected in plasma. We conclude that fetal and placental mRNA circulates in the blood of pregnant women. Transcriptional analysis of maternal whole blood identifies a unique set of biologically diverse fetal genes and has a multitude of clinical applications. [ABSTRACT FROM AUTHOR]

Background Genetic heritability and maternal atopy have been correlated to antenatal IgE production, but very few studies have studied gene–maternal atopy interaction on antenatal IgE production. This study investigated the interaction of CTLA-4 polymorphism with prenatal factors on the elevation of cord blood IgE (CBIgE). Methods Pregnant women were antenatally recruited for collection of prenatal environmental factors by a questionnaire. Umbilical cord blood samples were collected for CBIgE detection by fluorescence-linked enzyme assay and CTLA-4 polymorphism measurement by restriction fragment length polymorphism. Results A total of 1104 pregnant women initially participated in this cohort study, and 898 of them completed cord blood collection. 21.4% of the newborns had elevation of CBIgE (⩾0.5 kU/L). The CTLA-4+49A allele ( P=0.021), maternal atopy ( P<0.001) and gender ( P=0.034), but not the CTLA-4+49G allele, −318C allele, −318T allele, parental smoking or paternal atopy, were significantly correlated with the CBIgE elevation in multivariate analysis. A dichotomous analysis of gene–maternal atopy interactions identified maternal atopy and CTLA-4+49A allele had an additive effect on the CBIgE elevation, especially prominent in male newborns; and in the absence of maternal atopy, CTLA-4+49GG genotype had a protective effect on CBIgE elevation in female newborns. Conclusions Maternal but not paternal atopy has significant impacts on CBIgE elevation depending on gender and CTLA-4+49A/G polymorphism of newborns. Control of maternal atopy and modulation of CTLA-4 expression in the prenatal stage may be a target for the early prevention of perinatal allergy sensitization. [ABSTRACT FROM AUTHOR]

Abstract: The literature on whole genome amplification (WGA) techniques and their application to preimplantation genetic diagnosis (PGD) and prenatal diagnosis is reviewed. General polymerase chain reaction (PCR) fails to provide adequate information from limited cells in PGD and non-invasive prenatal diagnosis. Therefore several WGA techniques, such as primer extension preamplification (PEP) and degenerate oligonucleotide primed PCR (DOP-PCR), have been developed and successfully applied to clinical work during the past decade, especially in PGD and prenatal diagnosis. These techniques can provide ample amplification of genetic sequences from single cells for a series of subsequent PCR analyses such as restriction fragment length polymorphisms (RFLP) and comparative genomic hybridization (CGH), thus opening up a new area for prenatal diagnosis. However, several problems have been reported in the application of these techniques. The ideal WGA technique should have high yield, faithful representation of the original template, complete coverage of the genome, and simply performed procedure. In order to make good use of these techniques in future research and clinical work, it is undoubtedly necessary for an extensive understanding of the merits and pitfalls of these recently developed techniques. [Copyright &y& Elsevier]