Genomic and molecular characterization of preterm birth.

Preterm birth (PTB) complications are the leading cause of long-term morbidity and mortality in children. By using whole blood samples, we integrated whole-genome sequencing (WGS), RNA sequencing (RNA-seq), and DNA methylation data for 270 PTB and 521 control families. We analyzed this combined dataset to identify genomic variants associated with PTB and secondary analyses to identify variants associated with very early PTB (VEPTB) as well as other subcategories of disease that may contribute to PTB. We identified differentially expressed genes (DEGs) and methylated genomic loci and performed expression and methylation quantitative trait loci analyses to link genomic variants to these expression and methylation changes. We performed enrichment tests to identify overlaps between new and known PTB candidate gene systems. We identified 160 significant genomic variants associated with PTB-related phenotypes. The most significant variants, DEGs, and differentially methylated loci were associated with VEPTB. Integration of all data types identified a set of 72 candidate biomarker genes for VEPTB, encompassing genes and those previously associated with PTB. Notably, PTB-associated genes RAB31 and RBPJ were identified by all three data types (WGS, RNA-seq, and methylation). Pathways associated with VEPTB include EGFR and prolactin signaling pathways, inflammation- and immunity-related pathways, chemokine signaling, IFN-γ signaling, and Notch1 signaling. Progress in identifying molecular components of a complex disease is aided by integrated analyses of multiple molecular data types and clinical data. With these data, and by stratifying PTB by subphenotype, we have identified associations between VEPTB and the underlying biology.

Comparison of Infant Gut and Skin Microbiota, Resistome and Virulome Between Neonatal Intensive Care Unit (NICU) Environments.

Background: There is a growing move to provide care for premature infants in a single family, private room neonatal intensive care unit (NICU) in place of the traditional shared space, open bay NICU. The resultant effect on the developing neonatal microbiota is unknown. Study Design: Stool and groin skin swabs were collected from infants in a shared-space NICU (old NICU) and a single-family room NICU (new NICU) on the same hospital campus. Metagenomic sequencing was performed and data analyzed by CosmosID bioinformatics software package. Results: There were no significant differences between the cohorts in gestational age, length of stay, and delivery mode; infants in the old NICU received significantly more antibiotics (p = 0.03). Differentially abundant antimicrobial resistance genes and virulence associated genes were found between the cohorts in stool and skin, with more differentially abundant antimicrobial resistance genes in the new NICU. The entire bacterial microbiota analyzed to the genus level significantly differed between cohorts in skin (p = 0.0001) but not in stool samples. There was no difference in alpha diversity between the two cohorts. DNA viruses and fungi were detected but did not differ between cohorts. Conclusion: Differences were seen in the resistome and virulome between the two cohorts with more differentially abundant antimicrobial resistance genes in the new NICU. This highlights the influence that different NICU environments can have on the neonatal microbiota. Whether the differences were due to the new NICU being a single-family NICU or located in a newly constructed building warrants exploration. Long term health outcomes from the differences observed must be followed longitudinally.

Gene-Centric Analysis of Preeclampsia Identifies Maternal Association at PLEKHG1.

The genetic susceptibility to preeclampsia, a pregnancy-specific complication with significant maternal and fetal morbidity, has been poorly characterized. To identify maternal genes associated with preeclampsia risk, we assembled 498 cases and 1864 controls of European ancestry from preeclampsia case-control collections in 5 different US sites (with additional matched population controls), genotyped samples on a cardiovascular gene-centric array composed of variants from ≈2000 genes selected based on prior genetic studies of cardiovascular and metabolic diseases and performed case-control genetic association analysis on 27 429 variants passing quality control. In silico replication testing of 9 lead signals with P<10-4 was performed in independent European samples from the SOPHIA (Study of Pregnancy Hypertension in Iowa) and Inova cohorts (212 cases, 456 controls). Multiethnic assessment of lead signals was then performed in samples of black (26 cases, 136 controls), Hispanic (132 cases, 468 controls), and East Asian (9 cases, 80 controls) ancestry. Multiethnic meta-analysis (877 cases, 3004 controls) revealed a study-wide statistically significant association of the rs9478812 variant in the pleiotropic PLEKHG1 gene (odds ratio, 1.40 [1.23-1.60]; Pmeta=5.90×10-7). The rs9478812 effect was even stronger in the subset of European cases with known early-onset preeclampsia (236 cases diagnosed <37 weeks, 1864 controls; odds ratio, 1.59 [1.27-1.98]; P=4.01×10-5). PLEKHG1 variants have previously been implicated in genome-wide association studies of blood pressure, body weight, and neurological disorders. Although larger studies are required to further define maternal preeclampsia heritability, this study identifies a novel maternal risk locus for further investigation.

Introducing personalized health for the family: the experience of a single hospital system.

Pharmacogenetic testing is leading the personalized health movement, gradually being implemented in a variety of healthcare settings. To inform the efforts of other hospital and clinical practices implementing personalized health or medicine applications, we describe the implementation of a newborn pharmacogenetic testing program at Inova Health System (VA, USA). In particular, we describe the efforts to gather patient feedback through focus groups, the training and program staff, the pilot program and our experiences to date. In our experience, a multidisciplinary team was essential to address the myriad facets of program development and implementation as well as an in-person approach to introduce testing and patient education.

Collection of non-meconium stool on fecal occult blood cards is an effective method for fecal microbiota studies in infants.

BACKGROUND: Effective methods are needed to collect fecal samples from children for large-scale microbiota studies. Stool collected on fecal occult blood test (FOBT) cards that can be mailed provides an effective solution; however, the quality of sequencing resulting from this method is unknown. The aim of this study is to compare microbiota metrics of 16S ribosomal RNA (rRNA) gene sequencing from stool and meconium collected on FOBT cards with stool collected in an Eppendorf tube (ET) under different conditions. METHODS: Eight stool samples from children in diapers aged 0 month-2 years and three meconium samples were collected and stored as follows: (1) ≤ 2 days at room temperature (RT) in an ET, (2) 7 days at - 80 °C in an ET, (3) 3-5 days at RT on a FOBT card, (4) 7 days at RT on a FOBT card, and (5) 7 days at - 80 °C on a FOBT card. Samples stored at - 80 °C were frozen immediately. Each specimen/condition underwent 16S rRNA gene sequencing with replicates on the Illumina MiSeq. Alpha and beta diversity measures and relative abundance of major phyla were compared between storage conditions and container (ET vs. FOBT card), with pairwise comparison between different storage conditions and the "standard" of 7 days at - 80 °C in an ET and fresh stool in an ET. RESULTS: Stool samples clustered mainly by individual diaper (P < 10-5, Adonis), rather than by storage condition (P = 0.42) or container (P = 0.16). However, meconium samples clustered more by container (P = 0.002) than by individual diaper (P = 0.009) and storage condition (P = 0.02). Additionally, there were no differences in alpha diversity measures and relative abundance of major phyla after Bonferroni correction between stool stored on a FOBT card at RT for 7 days with stool stored in an ET tube at - 80 °C; differences in alpha diversity were seen however when compared to fresh stool in an ET. Overall, based on the intraclass correlation coefficient (ICC), the different storage containers/conditions are reliable in preserving the microbial memberships and slightly less reliable in preserving the alpha diversity and relative microbial composition of infant stool. CONCLUSIONS: Acknowledging certain limitations, FOBT cards may be a useful tool in large-scale stool microbiota studies in children requiring outpatient follow-up where only small amounts of stool can be obtained, but should not be used when studying meconium.

Utilization of genomic sequencing for population screening of immunodeficiencies in the newborn.

PurposeImmunodeficiency screening has been added to many state-directed newborn screening programs. The current methodology is limited to screening for severe T-cell lymphopenia disorders. We evaluated the potential of genomic sequencing to augment current newborn screening for immunodeficiency, including identification of non-T cell disorders.MethodsWe analyzed whole-genome sequencing (WGS) and clinical data from a cohort of 1,349 newborn-parent trios by genotype-first and phenotype-first approaches. For the genotype-first approach, we analyzed predicted protein-impacting variants in 329 immunodeficiency-related genes in the WGS data. As a phenotype-first approach, electronic health records were used to identify children with clinical features suggestive of immunodeficiency. Genomes of these children and their parents were analyzed using a separate pipeline for identification of candidate pathogenic variants for rare Mendelian disorders.ResultsWGS provides adequate coverage for most known immunodeficiency-related genes. 13,476 distinct variants and 8,502 distinct predicted protein-impacting variants were identified in this cohort; five individuals carried potentially pathogenic variants requiring expert clinical correlation. One clinically asymptomatic individual was found genomically to have complement component 9 deficiency. Of the symptomatic children, one was molecularly identified as having an immunodeficiency condition and two were found to have other molecular diagnoses.ConclusionNeonatal genomic sequencing can potentially augment newborn screening for immunodeficiency.

The Microbiome in Necrotizing Enterocolitis: A Case Report in Twins and Minireview.

Our case describes the serial microbiome changes in twins discordant for necrotizing enterocolitis (NEC), who shared similar intrauterine and early environmental exposures. The key findings were that the 2 neonates had distinctly different microbiome compositions from the first stool samples collected. Also, in the twin who developed NEC there was a decrease in bacterial diversity and an increase in Proteobacteria a week before developing any clinical symptoms, suggesting an early role of the intestinal microbiome in the development of NEC. Here we briefly review the literature on the role of the intestinal microbiome in NEC and how a greater understanding of the neonatal microbiome and host interactions may help mitigate this devastating disease.

Utility of whole-genome sequencing for detection of newborn screening disorders in a population cohort of 1,696 neonates.

PURPOSE: To assess the potential of whole-genome sequencing (WGS) to replicate and augment results from conventional blood-based newborn screening (NBS). METHODS: Research-generated WGS data from an ancestrally diverse cohort of 1,696 infants and both parents of each infant were analyzed for variants in 163 genes involved in disorders included or under discussion for inclusion in US NBS programs. WGS results were compared with results from state NBS and related follow-up testing. RESULTS: NBS genes are generally well covered by WGS. There is a median of one (range: 0-6) database-annotated pathogenic variant in the NBS genes per infant. Results of WGS and NBS in detecting 28 state-screened disorders and four hemoglobin traits were concordant for 88.6% of true positives (n = 35) and 98.9% of true negatives (n = 45,757). Of the five infants affected with a state-screened disorder, WGS identified two whereas NBS detected four. WGS yielded fewer false positives than NBS (0.037 vs. 0.17%) but more results of uncertain significance (0.90 vs. 0.013%). CONCLUSION: WGS may help rule in and rule out NBS disorders, pinpoint molecular diagnoses, and detect conditions not amenable to current NBS assays.

Mitotic stability of small supernumerary marker chromosomes depends on their shape and telomeres - a long term in vitro study.

Mosaicism is present in more than 50% of the cases with small supernumerary marker chromosomes (sSMCs) and karyotype 47,XX,+mar/46,XX or 47,XY,+mar/46,XY. Recently we provided first evidence that the mitotic stability of sSMC is dependent on their structure, i.e. their shape. Thus, here we performed a long term in vitro study on 12 selected cell lines from the Else Kröner-Fresenius-sSMC-cellbank (http://ssmc-tl.com/ekf-cellbank.html) to test mitotic sSMC stability systematically. The obtained results showed that inverted duplicated shaped and also the so-called complex sSMCs (group 1) are by far more stable, than centric-minute- or ring-shaped sSMCs (groups 2). Generally speaking, the percentage of cells with group-1-sSMCs remained stable over 90 days of cell culture, while that of group-2-sSMCs in parts dramatically decreased. In one group-2-cell line the sSMC was even lost completely after 30 days of in vitro culture, in others the sSMC was depleted in up to 40% of the cells. Still the highest rate of sSMC loss was recorded during EBV-transformation. Overall, the major difference between groups 1 and 2 was the number of telomeres per sSMC. In group 1 the sSMCs had "original" telomeres at both of their ends; in group 2 the sSMCs had either no, possibly secondary acquired and/or only one original telomere. This absence of protective telomeric sequences in group 2 seems to make sSMC more susceptible for loss during cell division. Still, also a growth advantage of cells without sSMC cannot be neglected entirely.

The increasing role of genetics and genomics in women's health.

Genetic and genomic testing are a clinical reality in health care today. Persons at risk for disease or who are simply curious about their genomes can have them analyzed. An individual's genome is a function of ancestry, family history and personal health and environmental exposures. Clinical and pharmacologic information can be obtained through genomic analysis. Genomic testing can be done by health care providers but some results can now be obtained through direct-to-consumer tests. Many ethical questions are being raised regarding genomic testing. Nurses can provide more optimal care by understanding the process of genomic testing as well as the implications of the results.

Mitotic stability of small supernumerary marker chromosomes: a study based on 93 immortalized cell lines.

Small supernumerary marker chromosomes (sSMC) are known for being present in mosaic form as 47,+mar/46 in >50% of the cases with this kind of extra chromosomes. However, no detailed studies have been done for the mitotic stability of sSMC so far, mainly due to the lack of a corresponding in vitro model system. Recently, we established an sSMC-cell bank (Else Kröner-Fresenius-sSMC-cellbank) with >150 cell lines. Therefore, 93 selected sSMC cases were studied here for the presence of the corresponding marker chromosomes before and after Epstein-Barr virus-induced immortalization. The obtained results showed that dicentric inverted duplicated-shaped sSMC are by far more stable in vitro than monocentric centric minute- or ring-shaped sSMC. Simultaneously, a review of the literature revealed that a comparable shape-dependent mitotic stability can be found in vivo in sSMC carriers. Additionally, a possible impact of the age of the sSMC carrier on mitotic stability was found: sSMC cell lines established from patients between 10-20 years of age were predominantly mitotically unstable. The latter finding was independent of the sSMC shape. The present study shows that in vitro models can lead to new and exciting insights into the biology of this genetically and clinically heterogeneous patient group.

Complex small supernumerary marker chromosomes - an update.

BACKGROUND: Complex small supernumerary marker chromosomes (sSMC) constitute one of the smallest subgroups of sSMC in general. Complex sSMC consist of chromosomal material derived from more than one chromosome; the best known representative of this group is the derivative chromosome 22 {der(22)t(11;22)} or Emanuel syndrome. In 2008 we speculated that complex sSMC could be part of an underestimated entity. RESULTS: Here, the overall yet reported 412 complex sSMC are summarized. They constitute 8.4% of all yet in detail characterized sSMC cases. The majority of the complex sSMC is contributed by patients suffering from Emanuel syndrome (82%). Besides there are a der(22)t(8;22)(q24.1;q11.1) and a der(13)t(13;18)(q11;p11.21) or der(21)t(18;21)(p11.21;q11.1) = der(13 or 21)t(13 or 21;18) syndrome. The latter two represent another 2.6% and 2.2% of the complex sSMC-cases, respectively. The large majority of complex sSMC has a centric minute shape and derives from an acrocentric chromosome. Nonetheless, complex sSMC can involve material from each chromosomal origin. Most complex sSMC are inherited form a balanced translocation in one parent and are non-mosaic. Interestingly, there are hot spots for the chromosomal breakpoints involved. CONCLUSIONS: Complex sSMC need to be considered in diagnostics, especially in non-mosaic, centric minute shaped sSMC. As yet three complex-sSMC-associated syndromes are identified. As recurrent breakpoints in the complex sSMC were characterized, it is to be expected that more syndromes are identified in this subgroup of sSMC. Overall, complex sSMC emphasize once more the importance of detailed cytogenetic analyses, especially in patients with idiopathic mental retardation.

Multiple copies of BCR-ABL fusion gene on two isodicentric Philadelphia chromosomes in an imatinib mesylate-resistant chronic myeloid leukemia patient.

The so-called Philadelphia (Ph) chromosome is present in more than 90% of chronic myeloid leukemia (CML) cases. Amplification or duplication of the BCR-ABL gene has been found to be one of the key factors leading to drug resistance to imatinib mesylate (IM). In the present study, we identified the presence of isodicentric Ph chromosomes [idic(Ph)] in an IM-resistant patient. Fluorescence in situ hybridization (FISH) analysis on metaphase chromosomes confirmed the heterogeneity and amplification of the fused BCR-ABL gene. FISH analysis superimposed on G-banding confirmed the presence of idic(Ph) chromosomes. Reverse transcription-polymerase chain reaction (RT-PCR) products revealed the presence of the BCR-ABL fusion transcript b3a2. The idic(Ph) chromosomes in CML were shown to be fused at the satellite regions of the short arms. The patient did not respond to IM chemotherapy and did not achieve remission. In this study, the impact of the idic(Ph) chromosomes on genomic instability, heterogeneity and amplification of the BCR-ABL gene in IM-resistant patients is discussed.

Multicolor FISH methods in current clinical diagnostics.

Multicolor FISH (mFISH) assays are currently indispensable for a precise description of derivative chromosomes. Routine application of such techniques on human chromosomes started in 1996 with the simultaneous use of all 24 human whole-chromosome painting probes in multiplex-FISH and spectral karyotyping. Since then, multiple approaches for chromosomal differentiation based on multicolor-FISH (MFISH) assays have been developed. Predominantly, they are applied to characterize marker or derivative chromosomes identified in conventional banding analysis. Since the introduction of array-based comparative genomic hybridization (aCGH), mFISH is also applied to verify and further delineate aCGH-detected aberrations. For the latter, it is important to consider the fact that aCGH cannot detect or characterize balanced rearrangements, which are important to be resolved in detail in infertility diagnostics. In addition, mFISH is necessary to distinguish different imbalanced situations detectable in aCGH; small supernumerary marker chromosomes have to be differentiated from insertions or unbalanced translocations. This review presents an overview on the available mFISH methods and their applications in pre- and post-natal clinical genetics.

Characterization of three small supernumerary marker chromosomes (sSMC) in humans.

In the present study, three prenatally detected small supernumerary marker chromosomes (sSMC) were identified by banding cytogenetics and characterized in detail by molecular cytogenetics. In one case an sSMC(10) leading to a pericentric partial trisomy and in two cases heterochromatic sSMC derived from chromosome 22 were characterized. Outcomes were reportedly normal for two of the three cases for whom this information was known.

Human ring chromosomes and small supernumerary marker chromosomes-do they have telomeres?

Ring chromosomes and small supernumerary marker chromosomes (sSMC) are enigmatic types of derivative chromosomes, in which the telomeres are thought to play a crucial role in their formation and stabilization. Considering that there are only a few studies that evaluate the presence of telomeric sequences in ring chromosomes and on sSMC, here, we analyzed 14 ring chromosomes and 29 sSMC for the presence of telomeric sequences through fluorescence in situ hybridization (FISH). The results showed that ring chromosomes can actually fall into two groups: the ones with or without telomeres. Additionally, telomeric signals were detectable at both ends of centric and neocentric sSMC with inverted duplication shape, as well as in complex sSMC. Apart from that, generally both ring- and centric minute-shaped sSMC did not present telomeric sequences neither detectable by FISH nor by a second protein-directed immunohistochemical approach. However, the fact that telomeres are absent does not automatically mean that the sSMC has a ring shape, as often deduced in the previous literature. Overall, the results obtained by FISH studies directed against telomeres need to be checked carefully by other approaches.

Detailed analysis of an idic(Y)(q11.21) in a mosaic karyotype.

Abnormalities involving sex chromosomes account for approximately 0.5% of live births. The phenotypes of individuals with mosaic cell lines that exhibit structural aberrations of the X and Y chromosomes are variable and difficult to predict. Phenotypes associated with sex chromosome mosaicism vary from females with Turner syndrome to males with infertility, and include individuals with ambiguous genitalia. In this study, we report a 17-year-old male with phenotypic features of Klinefelter syndrome with an isodicentric Y chromosome and a final karyotype of 45,X[4]/46,X,idic(Y)(q11.21)[95]/47,XX,+idic(Y)(q11.21)[1]. Application of high resolution molecular cytogenetic techniques as well as molecular studies revealed two copies of the sex-determining region of Y chromosome (SRY) gene and two centromers. Additionally, the breakpoint in Yq11.21 was narrowed down between positions 13.4 and 14.3 MB (hg18). We present a patient with partial disomy of Ypter to Yq11.21 in the majority of the patient cells, showing phenotypic features of Klinefelter syndrome. The syndrome may have occurred due to a more prominent presence of the cell line 47,XX,+idic(Y)(q11.21) detected only once in 1% of the peripheral blood cells. This finding may prove helpful in similar cases with symptoms of Klinefelter syndrome, but which exhibit an absence of the cell line 47,XXY in peripheral blood.

How to narrow down chromosomal breakpoints in small and large derivative chromosomes--a new probe set.

Here a new fluorescence in situ hybridization (FISH-) based probe set is presented and its possible applications are highlighted in 34 exemplary clinical cases. The so-called pericentric-ladder-FISH (PCL-FISH) probe set enables a characterization of chromosomal breakpoints especially in small supernumerary marker chromosomes (sSMC), but can also be applied successfully in large inborn or acquired derivative chromosomes. PCL-FISH was established as 24 different chromosome-specific probe sets and can be used in two- up multicolor-FISH approaches. PCL-FISH enables the determination of a chromosomal breakpoint with a resolution between 1 and ∼10 megabasepairs and is based on locus-specific bacterial artificial chromosome (BAC) probes. Results obtained on 29 sSMC cases and five larger derivative chromosomes are presented and discussed. To confirm the reliability of PCL-FISH, eight of the 29 sSMC cases were studied by array-comparative genomic hybridization (aCGH); the used sSMC-specific DNA was obtained by glass-needle based microdissection and DOP-PCR-amplification. Overall, PCL-FISH leads to a better resolution than most FISH-banding approaches and is a good tool to narrow down chromosomal breakpoints.

Microdeletion and microduplication syndromes.

The widespread use of whole genome analysis based on array comparative genomic hybridization in diagnostics and research has led to a continuously growing number of microdeletion and microduplication syndromes (MMSs) connected to certain phenotypes. These MMSs also include increasing instances in which the critical region can be reciprocally deleted or duplicated. This review catalogues the currently known MMSs and the corresponding critical regions including phenotypic consequences. Besides the pathogenic pathways leading to such rearrangements, the different detection methods and their limitations are discussed. Finally, the databases available for distinguishing between reported benign or pathogenic copy number alterations are highlighted. Overall, a review of MMSs that previously were also denoted "genomic disorders" or "contiguous gene syndromes" is given.

Centromeric association of small supernumerary marker chromosomes with their sister-chromosomes detected by three dimensional molecular cytogenetics.

BACKGROUND: Small supernumerary marker chromosomes (sSMC) are detected in 0.043% of general population and can be characterized for their chromosomal origin, genetic content and shape by molecular cytogenetic approaches. Even though recently progress was achieved towards genotype-phenotype-correlations of sSMC, nothing is known on the influence that an additional derivative extra chromosome has on the nuclear architecture. RESULTS: Here we present the first three-dimensional interphase fluorescence in situ hybridization (FISH) studies for the nuclear architecture of sSMC. It could be shown that sSMC derived from chromosomes 15, 16 or 18 preferentially colocalized with one of their corresponding sister chromosomes. This was true in B- and T-lymphocytes as well as in skin fibroblasts. Additionally, a case with a complex sSMC with a karyotype 47,XY,+der(18)t(8;18)(8p23.2 ~ 23.1;18q11.1) was studied. Here the sSMC co-localized with one homologous chromosome 8 instead of 18. CONCLUSION: Overall, there is a kind of "attraction" between an sSMC and one of its homologous sister chromosomes. This seems to be transmitted by the euchromatic part of the sSMC rather than its heterochromatic one.