Ying Sheng

Limitations and possibilities of low cell number ChIP-seq

BackgroundChromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq) offers high resolution, genome-wide analysis of DNA-protein interactions. However, current standard methods require abundant starting material in the range of 1–20 million cells per immunoprecipitation, and remain a bottleneck to the acquisition of biologically relevant epigenetic data. Using a ChIP-seq protocol optimised for low cell numbers (down to 100,000 cells / IP), we examined the performance of the ChIP-seq technique on a series of decreasing cell numbers.ResultsWe present an enhanced native ChIP-seq method tailored to low cell numbers that represents a 200-fold reduction in input requirements over existing protocols. The protocol was tested over a range of starting cell numbers covering three orders of magnitude, enabling determination of the lower limit of the technique. At low input cell numbers, increased levels of unmapped and duplicate reads reduce the number of unique reads generated, and can drive up sequencing costs and affect sensitivity if ChIP is attempted from too few cells.ConclusionsThe optimised method presented here considerably reduces the input requirements for performing native ChIP-seq. It extends the applicability of the technique to isolated primary cells and rare cell populations (e.g. biobank samples, stem cells), and in many cases will alleviate the need for cell culture and any associated alteration of epigenetic marks. However, this study highlights a challenge inherent to ChIP-seq from low cell numbers: as cell input numbers fall, levels of unmapped sequence reads and PCR-generated duplicate reads rise. We discuss a number of solutions to overcome the effects of reducing cell number that may aid further improvements to ChIP performance.

Primary immunodeficiency diseases: Genomic approaches delineate heterogeneous Mendelian disorders

Background: Primary immunodeficiency diseases (PIDDs) are clinically and genetically heterogeneous disorders thus far associated with mutations in more than 300 genes. The clinical phenotypes derived from distinct genotypes can overlap. Genetic etiology can be a prognostic indicator of disease severity and can influence treatment decisions. Objective: We sought to investigate the ability of whole‐exome screening methods to detect disease‐causing variants in patients with PIDDs. Methods: Patients with PIDDs from 278 families from 22 countries were investigated by using whole‐exome sequencing. Computational copy number variant (CNV) prediction pipelines and an exome‐tiling chromosomal microarray were also applied to identify intragenic CNVs. Analytic approaches initially focused on 475 known or candidate PIDD genes but were nonexclusive and further tailored based on clinical data, family history, and immunophenotyping. Results: A likely molecular diagnosis was achieved in 110 (40%) unrelated probands. Clinical diagnosis was revised in about half (60/110) and management was directly altered in nearly a quarter (26/110) of families based on molecular findings. Twelve PIDD‐causing CNVs were detected, including 7 smaller than 30 Kb that would not have been detected with conventional diagnostic CNV arrays. Conclusion: This high‐throughput genomic approach enabled detection of disease‐related variants in unexpected genes; permitted detection of low‐grade constitutional, somatic, and revertant mosaicism; and provided evidence of a mutational burden in mixed PIDD immunophenotypes.

Identification of copy number variants from exome sequence data

BackgroundWith advances in next generation sequencing technologies and genomic capture techniques, exome sequencing has become a cost-effective approach for mutation detection in genetic diseases. However, computational prediction of copy number variants (CNVs) from exome sequence data is a challenging task. Whilst numerous programs are available, they have different sensitivities, and have low sensitivity to detect smaller CNVs (1–4 exons). Additionally, exonic CNV discovery using standard aCGH has limitations due to the low probe density over exonic regions. The goal of our study was to develop a protocol to detect exonic CNVs (including shorter CNVs that cover 1–4 exons), combining computational prediction algorithms and a high-resolution custom CGH array.ResultsWe used six published CNV prediction programs (ExomeCNV, CONTRA, ExomeCopy, ExomeDepth, CoNIFER, XHMM) and an in-house modification to ExomeCopy and ExomeDepth (ExCopyDepth) for computational CNV prediction on 30 exomes from the 1000 genomes project and 9 exomes from primary immunodeficiency patients. CNV predictions were tested using a custom CGH array designed to capture all exons (exaCGH). After this validation, we next evaluated the computational prediction of shorter CNVs. ExomeCopy and the in-house modified algorithm, ExCopyDepth, showed the highest capability in detecting shorter CNVs. Finally, the performance of each computational program was assessed by calculating the sensitivity and false positive rate.ConclusionsIn this paper, we assessed the ability of 6 computational programs to predict CNVs, focussing on short (1–4 exon) CNVs. We also tested these predictions using a custom array targeting exons. Based on these results, we propose a protocol to identify and confirm shorter exonic CNVs combining computational prediction algorithms and custom aCGH experiments.

Placental miR-1301 is dysregulated in early-onset preeclampsia and inversely correlated with maternal circulating leptin.

INTRODUCTION miRNAs are small non-coding RNAs important for the regulation of mRNA in many organs including placenta. Adipokines and specifically leptin are known to be dysregulated in preeclampsia, but little is known regarding their regulation by miRNAs during pregnancy. METHODS We performed high-throughput sequencing of small RNAs in placenta from 72 well-defined patients: 23 early-onset preeclampsia (PE), 26 late-onset PE and 23 controls. The regulation of some miRNAs was confirmed on qRT-PCR. Maternal circulating levels and placental mRNA of leptin, resistin and adiponectin were measured using Bio-Plex and qRT-PCR. RESULTS We found that miR-1301, miR-223 and miR-224 expression was downregulated in early-onset PE, but not in late-onset PE, compared to controls. In silico analysis predicted the leptin gene (LEP) to be a target for all three miRNAs. Indeed, we found significant correlation between maternal circulating levels of leptin and placental LEP expression. In addition, we found a significant inverse correlation between maternal circulating leptin/placental LEP expression and placental miR-1301 expression levels. Interestingly, placental expression of miR-1301 was also correlated with newborn weight percentile and inversely correlated with both maternal systolic and diastolic blood pressure prior to delivery. DISCUSSION Our results confirm that placenta is a major site of LEP expression during pregnancy. It further suggests that miR-1301 could be involved in the regulation of leptin during pregnancy and may play a role in early-onset PE. CONCLUSIONS miR-1301 is dysregulated in early-onset preeclampsia and could possibly play a role in the regulation of leptin during pregnancy.

Strømme Syndrome Is a Ciliary Disorder Caused by Mutations in CENPF.

Strømme syndrome was first described by Strømme et al. (1993) in siblings presenting with “apple peel” type intestinal atresia, ocular anomalies and microcephaly. The etiology remains unknown to date. We describe the long‐term clinical follow‐up data for the original pair of siblings as well as two previously unreported siblings with a severe phenotype overlapping that of the Strømme syndrome including fetal autopsy results. Using family‐based whole‐exome sequencing, we identified truncating mutations in the centrosome gene CENPF in the two nonconsanguineous Caucasian sibling pairs. Compound heterozygous inheritance was confirmed in both families. Recently, mutations in this gene were shown to cause a fetal lethal phenotype, the phenotype and functional data being compatible with a human ciliopathy [Waters et al., ]. We show for the first time that Strømme syndrome is an autosomal‐recessive disease caused by mutations in CENPF that can result in a wide phenotypic spectrum.

A founder mutation p.H701P identified as a major cause of SPG7 in Norway

SPG7 is one of the most common forms of autosomal recessive hereditary spastic paraplegia. The phenotype has been shown to be heterogeneous, varying from a complex spastic ataxia to pure spastic paraplegia or pure ataxia. The aim of this study was to clinically and genetically characterize patients with SPG7 in Norway.

Generalized epilepsy in a family with basal ganglia calcifications and mutations in SLC20A2 and CHRNB2

BACKGROUND The genetic understanding of primary familial brain calcification (PFBC) has increased considerably in recent years due to the finding of causal genes like SLC20A2, PDGFRB and PDGFB. The phenotype of PFBC is complex and has as of yet been poorly delineated. The most common clinical presentations include movement disorders, cognitive symptoms and psychiatric conditions. We report a family including two sisters with brain calcifications due to a variant in SLC20A2 and generalized tonic-clonic seizures as the principal phenotypic trait. METHODS The affected siblings underwent whole exome sequencing and candidate variants and cosegregation in the family were validated by Sanger sequencing. RESULTS Both siblings and their asymptomatic father were heterozygous for a variant in SLC20A2. The siblings also had a variant in CHRNB2, a known epilepsy gene associated with autosomal dominant frontal lobe epilepsy, which they had inherited from the mother. CONCLUSIONS To our knowledge, the reported siblings represent the third and fourth subjects with confirmed SLC20A2 variants exhibiting epilepsy as a phenotypic trait. Our findings support seizures as part of the phenotypic spectrum of SLC20A2-related PFBC. However, the present phenotype may also result from additional genetic influence, such as the identified missense variant in CHRNB2.

Kaufman oculocerebrofacial syndrome in sisters with novel compound heterozygous mutation in UBE3B.

A pair of sisters was ascertained for multiple congenital defects, including marked craniofacial dysmorphisms with blepharophimosis, and severe psychomotor delay. Two novel compound heterozygous mutations in UBE3B were identified in both the sisters by exome sequencing. These mutations include c.1A>G, which predicts p.Met1?, and a c.1773delC variant, predicted to cause a frameshift at p.Phe591fs. UBE3B encodes a widely expressed protein ubiquitin ligase E3B, which, when mutated in both alleles, causes Kaufman oculocerebrofacial syndrome. We report on the thorough clinical examination of the patients and review the state of art knowledge of this disorder.

Homozygous KIDINS220 loss-of-function variants in fetuses with cerebral ventriculomegaly and limb contractures

Heterozygous mutations in KIDINS220 were recently suggested a cause of spastic paraplegia, intellectual disability, nystagmus and obesity. All patients carried terminal nonsense de novo mutations that seemed to escape nonsense-mediated mRNA decay. The mechanism for pathogenicity is yet unexplained, as it seems that heterozygous loss-of-function variants of KIDINS220 are generally well tolerated. We present a consanguineous couple who experienced four pregnancy terminations due to repeated findings in the fetuses comprising enlarged cerebral ventricles and limb contractures. Exome sequencing in two of the aborted fetuses revealed a shared homozygous frameshift variant in exon 24 in KIDINS220. Sanger sequencing of the variant in available family members showed complete segregation with the affection status, resulting in a LOD score of 2.5 under an autozygous inheritance model. mRNA studies revealed destruction of the original splice site, resulting in an out-of-frame transcript and introduction of a premature termination codon in exon 25. Premature termination codons in this position are likely to cause activation of nonsense-mediated mRNA decay and result in complete absence of KIDINS220 protein in individuals homozygous for the variant. The phenotype of the presented fetuses overlaps with findings in functional studies of knockout Kidins220 mice embryos that are non-viable with enlarged cerebral ventricles. The human fetuses also exhibit several similarities to the milder phenotype described in patients with heterozygous KIDINS220 mutations. We hence propose that the identified homozygous loss-of-function variant in KIDINS220 causes the phenotype in the presented fetuses, and that this represents a hitherto undescribed severe autosomal recessive neurodevelopmental disorder.

Exome Sequencing Fails to Identify the Genetic Cause of Aicardi Syndrome

Aicardi syndrome (AS) is a well-characterized neurodevelopmental disorder with an unknown etiology. In this study, we performed whole-exome sequencing in 11 female patients with the diagnosis of AS, in order to identify the disease-causing gene. In particular, we focused on detecting variants in the X chromosome, including the analysis of variants with a low number of sequencing reads, in case of somatic mosaicism. For 2 of the patients, we also sequenced the exome of the parents to search for de novo mutations. We did not identify any genetic variants likely to be damaging. Only one single missense variant was identified by the de novo analyses of the 2 trios, and this was considered benign. The failure to identify a disease gene in this study may be due to technical limitations of our study design, including the possibility that the genetic aberration leading to AS is situated in a non-exonic region or that the mutation is somatic and not detectable by our approach. Alternatively, it is possible that AS is genetically heterogeneous and that 11 patients are not sufficient to reveal the causative genes. Future studies of AS should consider designs where also non-exonic regions are explored and apply a sequencing depth so that also low-grade somatic mosaicism can be detected.