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Abstract Our aim was to develop and apply a comprehensive noninvasive prenatal test (NIPT) by using high-coverage targeted next-generation sequencing to estimate fetal fraction, determine fetal sex, and detect trisomy and monogenic disease without parental genotype information. We analyzed 45 pregnancies, 40 mock samples, and eight mother-child pairs to generate 35 simulated datasets. Fetal fraction (FF) was estimated based on analysis of the single nucleotide polymorphism (SNP) allele fraction distribution. A Z-score was calculated for trisomy of chromosome 21 (T21), and fetal sex detection. Monogenic disease detection was performed through variant analysis. Model validation was performed using the simulated datasets. The novel model to estimate FF was robust and accurate (r2= 0.994, p-value < 2.2e-16). For samples with FF > 0.04, T21 detection had 100% sensitivity (95% CI: 63.06 to 100%) and 98.53% specificity (95% CI: 92.08 to 99.96%). Fetal sex was determined with 100% accuracy. We later performed a proof of concept for monogenic disease diagnosis of 5/7 skeletal dysplasia cases. In conclusion, it is feasible to perform a comprehensive NIPT by using only data from high coverage targeted sequencing, which, in addition to detecting trisomies, also make it possible to identify pathogenic variants of the candidate genes for monogenic diseases.

Despite the fact that patients homozygous for the sickle cell disease (SCD) mutation have an identical genotype, the severity of the disease can be extremely variable. The hemoglobin (Hb) S mutation has been described on five different haplotypes with different clinical expression. Identifying the genotypes, haplotypes and sub-haplotypes of the β gene cluster in Oman needs to be studied in more details to establish a correlation between the genotype/haplotype and phenotype diversity observed in SCD patients for prognostic purposes, accurate diagnosis and thus planning for the best tailored treatment. We have investigated 125 HbS homozygotes from different parts of Oman and determined their haplotypes and sub-haplotypes and correlated this to the hematological and clinical expression. We have found 11 haplotype combinations differently distributed in the country, with the Asian/Asian HbS haplotype being the most predominant. Sub-haplotypes was only found among patients with CAR/OmanI haplotype. As expected, the correlation between haplotypes, sub-haplotypes and disease severity was mainly associated with HbF expression. Our study on haplotype/phenotype correlation has shown which major haplotypes occur in the different regions of Oman. Furthermore, neither the haplotype or sub-haplotype nor the HbF alone appeared to be fully associable with the variable clinical phenotypes. External factors do occur and are associated with the expression of the disease. 尽管镰状细胞突变病（SCD）患者拥有相同的基因类型，但患者的病患程度却大相径庭。血红蛋白（Hb）S突变有五种不同的单体型，各种类型在临床表现上也不相同。为了识别在阿曼地区β基因簇的基因型、单体型，亚单体型，需要研究更多以SCD患者预后为目的，关于其观察到的基因型、单体型，表型多样性之间联系的更多细节，以便作出准确的诊断，为各个患者量身制定治疗方案。我们研究了125个来自阿曼不同地区的HbS纯合体，并确认了它们的单体型和亚单体型血液学上的临床表现。我们已找到了该国家11个单体型组合的不同分布，其中以亚洲/亚洲HbS单体型为主要类型。亚单体型只在CAR/OMANI单体型患者中被发现。正如之前所料，单体型、亚单体型和病患程度之间的联系主要与HbF表现相关。我们关于单体型和亚单体型之间联系的研究显示出了阿曼地区最为主要的单体类型。此外，无论是单体型、亚单体型还是HbF，都被证明与该疾病不同的临床表现没有紧密的联系。外部因素是该疾病不同表现的致因。

Hemoglobinopathies (HBP) are the most common genetic disorder in Oman and are in need of prevention programs due to the high incidence of β-thalassemia major and sickle cell disease. Prenatal diagnosis (PD) and selective pregnancy termination is shown to be the most effective prevention tool for the control of HBP. However, PD is not available in Oman thus far because abortion is subject to religious, cultural and ethical issues. We have examined the attitude of a number of Omani HBP carrier couples towards prenatal diagnosis and selective abortion. We have interviewed 35 couples at risk visiting the main premarital clinic in Muscat between Jan 2011 and Jan 2012. Couples were interviewed using a pre-structured questionnaire. The majority would have accepted prenatal diagnosis (94%) if the service would be available in the country but pregnancy termination was greatly influenced by religious values. 血红蛋白病（HBP）是一种在阿曼最常见的遗传性疾病，由于其高发的B型地中海贫血症及镰状细胞症，相关的预防措施对于这一国家来说，相当重要。产前诊断（PD）和选择性终止妊娠被证实是针对管控血红蛋白病（HBP）的最有效方法。然而，由于受到宗教、文化和伦理抵制堕胎的影响，产前诊断（PD）并不能在该国得以应用。我们对该国一部分血红蛋白病患夫妇做了一项关于产前诊断的意向调查。2011年一月至2012年一月，我们在马斯喀特（阿曼首都）的一家婚前诊所对35对夫妇做了相关的采访调查。调查的问卷是事先设置好的。大部分（94%）夫妇表示接受产前诊断如果相应的措施能得到广泛的普及，但是他们对于选择性终止妊娠的态度受到了其宗教价值观的极大影响。

The aims of this study are: i) to enquire whether informing healthy hemoglobinopathy carriers about their condition is a welcome initiative in The Netherlands; ii) to study whether using information letters and thorough explanation is associated with presence or absence of undesired feelings or emotions. We have approached 100 multi-ethnic carriers previously diagnosed in our lab. All subjects had previously received our information letter through their physician who was supposed to have provided an explanation of the letter if required. We have enquired whether the subjects had experienced negative or positive emotions after receiving our diagnosis and explanation and to which degree, if they were sufficiently informed and satisfied and if they would have considered prevention in case of risk. The rate negative versus positive feelings was calculated using a numerical distribution. We have registered negative feelings in a rate that was directly proportional to the lack of information. While the number of registered negative feelings in well-informed carriers was very low it was more present in badly informed. Nevertheless, all participants found carrier information a welcome initiative and over 80% of them declared to be in favor of prenatal diagnosis in case of risk.

Context. Leri–Weill dyschondrosteosis is a clinically variable skeletal dysplasia, caused by SHOX deletion or mutations, or a deletion of enhancer sequences in the 3’-flanking region. Recently, a 47.5 kb recurrent PAR1 deletion downstream of SHOX was reported, but its frequency and clinical importance are still unknown.Objective. This study aims to compare the clinical features of different sizes of deletions in the 3’-flanking SHOX region in order to determine the relevance of the regulatory sequences in this region.Design. We collected DNA from 28 families with deletions in the 3’-PAR1 region. Clinical data were available from 23 index patients and 21 relatives.Results. In 9 families (20 individuals) a large deletion ( ∼ 200–900 kb) was found and in 19 families (35 individuals) a small deletion was demonstrated, equal to the recently described 47.5 kb PAR1 deletion. Median height SDS, sitting height/height ratio SDS and the presence of Madelung deformity in patients with the 47.5 kb deletion were not significantly different from patients with larger deletions. The index patients had a median height SDS which was slightly lower than in their affected family members (p = 0.08). No significant differences were observed between male and female patients.Conclusions. The phenotype of patients with deletions in the 3’-PAR1 region is remarkably variable. Height, sitting height/height ratio and the presence of Madelung deformity were not significantly different between patients with the 47.5 kb recurrent PAR1 deletion and those with larger deletions, suggesting that this enhancer plays an important role in SHOX expression.

OBJECTIVES: RASSF1A has been described to be differentially methylated between fetal and maternal DNA and can therefore be used as a universal sex-independent marker to confirm the presence of fetal sequences in maternal plasma. However, this requires highly sensitive methods. We have previously shown that Pyrophosphorolysis-activated Polymerization (PAP) is a highly sensitive technique that can be used in noninvasive prenatal diagnosis. In this study, we have used PAP in combination with bisulfite conversion to develop a new universal methylation-based assay for the detection of fetal methylated RASSF1A sequences in maternal plasma. METHODS: Bisulfite sequencing was performed on maternal genomic (g)DNA and fetal gDNA from chorionic villi to determine differentially methylated regions in the RASSF1A gene using bisulfite specific PCR primers. Methylation specific primers for PAP were designed for the detection of fetal methylated RASSF1A sequences after bisulfite conversion and validated. RESULTS: Serial dilutions of fetal gDNA in a background of maternal gDNA show a relative percentage of ~3% can be detected using this assay. Furthermore, fetal methylated RASSF1A sequences were detected both retrospectively as well as prospectively in all maternal plasma samples tested (n = 71). No methylated RASSF1A specific bands were observed in corresponding maternal gDNA. Specificity was further determined by testing anonymized plasma from non-pregnant females (n = 24) and males (n = 21). Also, no methylated RASSF1A sequences were detected here, showing this assay is very specific for methylated fetal DNA. Combining all samples and controls, we obtain an overall sensitivity and specificity of 100% (95% CI 98.4%-100%). CONCLUSIONS: Our data demonstrate that using a combination of bisulfite conversion and PAP fetal methylated RASSF1A sequences can be detected with extreme sensitivity in a universal and sex-independent manner. Therefore, this assay could be of great value as an addition to current techniques used in noninvasive prenatal diagnostics.

ObjectiveFour types of antipsychotic-induced movement disorders: tardive dyskinesia (TD), parkinsonism, akathisia and tardive dystonia, subtypes of TD (orofacial and limb truncal dyskinesia), subtypes of parkinsonism (rest tremor, rigidity, and bradykinesia), as well as a principal-factor of the movement disorders and their subtypes, were examined for association with variation in 10 candidate genes (PPP1R1B, BDNF, DRD3, DRD2, HTR2A, HTR2C, COMT, MnSOD, CYP1A2, and RGS2).MethodsNaturalistic study of 168 white long-stay patients with chronic mental illness requiring long-term antipsychotic treatment, examined by the same rater at least two times over a 4-year period, with a mean follow-up time of 1.1 years, with validated scales for TD, parkinsonism, akathisia, and tardive dystonia. The authors genotyped 31 SNPs, associated with movement disorders or schizophrenia in previous studies. Genotype and allele frequency comparisons were performed with multiple regression methods for continuous movement disorders.ResultsVARIOUS SNPS REACHED NOMINAL SIGNIFICANCE: TD and orofacial dyskinesia with rs6265 and rs988748, limb truncal dyskinesia with rs6314, rest tremor with rs6275, rigidity with rs6265 and rs4680, bradykinesia with rs4795390, akathisia with rs4680, tardive dystonia with rs1799732, rs4880 and rs1152746. After controlling for multiple testing, no significant results remained.ConclusionsThe findings suggest that selected SNPs are not associated with a susceptibility to movement disorders. However, as the sample size was small and previous studies show inconsistent results, definite conclusions cannot be made. Replication is needed in larger study samples, preferably in longitudinal studies which take the fluctuating course of movement disorders and gene-environment interactions into account.

Duchenne and Becker muscular dystrophy (DMD and BMD) are caused by mutations in the dystrophin gene. Large rearrangements in the gene are found in about two-thirds of DMD patients, with ~60% carrying deletions and 5–10% carrying duplications. Most of the remaining 30–35% of patients are expected to have small nucleotide substitutions, insertions, or deletions. To detect these subtle changes within the coding and splice site determining sequences of the dystrophin gene, we established a semiautomated denaturing gradient gel electrophoresis (DGGE) mutation scanning system. The DGGE scan covers the dystrophin gene with 95 amplicons, PCRed either individually or in a multiplex setup. PCR and pooling were performed semiautomatically, using a pipetting robot and 384-well plates, enabling concurrent amplification of DNA of four patients in one run. Amplification of individual fragments was performed using one PCR program. The products were pooled just before gel loading; DGGE requires only a single gel condition. Validation was performed using DNA samples harboring 39 known DMD variants, all of which could be readily detected. DGGE mutation scanning was applied to analyze 135 DMD/BMD patients and potential DMD carriers without large deletions or duplications. In DNA from 25 out of 44 DMD patients (57%) and from 5 out of 39 BMD patients (13%), we identified clear pathogenic changes. All mutations were different, with the exception of one DMD mutation, which occurred twice. In DNA from 10 out of 44 potential DMD carriers, including four obligate carriers, we detected causative changes, including one pathogenic change in every obligate carrier. In addition to these pathogenic changes, we detected 15 unique unclassified variants, i.e., changes for which a pathogenic nature is uncertain. Hum Mutat 23:57–66, 2004. © 2003 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]