Clinical exome sequencing uncovers genetic disorders in neonates with suspected hypoxic-ischemic encephalopathy: A retrospective analysis.

Hypoxic-ischemic encephalopathy (HIE) occurs in up to 7 out of 1000 births and accounts for almost a quarter of neonatal deaths worldwide. Despite the name, many newborns with HIE have little evidence of perinatal hypoxia. We hypothesized that some infants with HIE have genetic disorders that resemble encephalopathy. We reviewed genetic results for newborns with HIE undergoing exome or genome sequencing at a clinical laboratory (2014-2022). Neonates were included if they had a diagnosis of HIE and were delivered ≥35 weeks. Neonates were excluded for cardiopulmonary pathology resulting in hypoxemia or if neuroimaging suggested postnatal hypoxic-ischemic injury. Of 24 patients meeting inclusion criteria, six (25%) were diagnosed with a genetic condition. Four neonates had variants at loci linked to conditions with phenotypic features resembling HIE, including KIF1A, GBE1, ACTA1, and a 15q13.3 deletion. Two additional neonates had variants in genes not previously associated with encephalopathy, including DUOX2 and PTPN11. Of the six neonates with a molecular diagnosis, two had isolated HIE without apparent comorbidities to suggest a genetic disorder. Genetic diagnoses were identified among neonates with and without sentinel labor events, abnormal umbilical cord gasses, and low Apgar scores. These results suggest that genetic evaluation is clinically relevant for patients with perinatal HIE.

Improving access to exome sequencing in a medically underserved population through the Texome Project.

PURPOSE: Genomic medicine can end diagnostic odysseys for patients with complex phenotypes; however, limitations in insurance coverage and other systemic barriers preclude individuals from accessing comprehensive genetics evaluation and testing. METHODS: The Texome Project is a 4-year study that reduces barriers to genomic testing for individuals from underserved and underrepresented populations. Participants with undiagnosed, rare diseases who have financial barriers to obtaining exome sequencing (ES) clinically are enrolled in the Texome Project. RESULTS: We highlight the Texome Project process and describe the outcomes of the first 60 ES results for study participants. Participants received a genetic evaluation, ES, and return of results at no cost. We summarize the psychosocial or medical implications of these genetic diagnoses. Thus far, ES provided molecular diagnoses for 18 out of 60 (30%) of Texome participants. Plus, in 11 out of 60 (18%) participants, a partial or probable diagnosis was identified. Overall, 5 participants had a change in medical management. CONCLUSION: To date, the Texome Project has recruited a racially, ethnically, and socioeconomically diverse cohort. The diagnostic rate and medical impact in this cohort support the need for expanded access to genetic testing and services. The Texome Project will continue reducing barriers to genomic care throughout the future study years.

Novel hemizygous single-nucleotide duplication in RPGR in a patient with retinal dystrophy and sensorineural hearing loss.

BACKGROUND: The RPGR gene has been associated with X-linked cone-rod dystrophy. This report describes a variant in RPGR detected with exome sequencing (ES). Genes like RPGR have not always been included in panel-based testing and thus genome-wide tests such as ES may be required for accurate diagnosis. METHODS: The Texome Project is studying the impact of ES in medically underserved patients who are in need of genomic testing to guide diagnosis and medical management. The hypothesis is that ES could uncover diagnoses not made by standard medical care. RESULTS: A 58-year-old male presented with retinitis pigmentosa, sensorineural hearing loss, and a family history of retinal diseases. A previous targeted gene panel for retinal disorders had not identified a molecular cause. ES through the Texome Project identified a novel, hemizygous variant in RPGR (NM_000328.3: c.1302dup, p.L435Sfs*18) that explained the ocular phenotype. CONCLUSIONS: Continued genetics evaluation can help to end diagnostic odysseys of patients. Careful consideration of genes represented when utilizing gene panels is crucial to ensure an accurate diagnosis. Medically underserved populations are less likely to receive comprehensive genetic testing in their diagnostic workup. Our report is an example of the medical impact of genomic medicine implementation.

A familial deletion of 10p12.1 associated with thrombocytopenia.

Thrombocytopenia can be inherited or acquired from a variety of causes. While hereditary causes of thrombocytopenia are rare, several genes have been associated with the condition. In this report, we describe an 18-year-old man and his mother, both of whom have congenital thrombocytopenia. Exome sequencing in the man revealed a 1006 kb maternally inherited deletion in the 10p12.1 region (arr[GRCh37] 10p12.1(27378928_28384564)x1) of uncertain clinical significance. This deletion in the THC2 locus includes genes ANKRD26, known to be involved in normal megakaryocyte differentiation, and MASTL, which some studies suggest is linked to autosomal dominant thrombocytopenia. In the family presented here, the deletion segregated with the congenital thrombocytopenia phenotype, suggesting that haploinsufficiency of one or both genes may be the cause. To our knowledge, this is the first report of a deletion of the THC2 locus associated with thrombocytopenia. Future functional studies of deletions of the THC2 locus may elucidate the mechanism for this phenotype observed clinically.

Precision therapy for a medically actionable ATP1A3 variant from a genomic medicine program in an underserved population.

BACKGROUND: Genomic medicine is revolutionizing the diagnosis of rare diseases, but the implementation has not benefited underrepresented populations to the same degree. Here, we report the case of a 7-year-old boy with hypotonia, global developmental delay, strabismus, seizures, and previously suspected mitochondrial myopathy. This proband comes from an underrepresented minority and was denied exome sequencing by his public insurance. METHODS: After informed consent was obtained, buccal cells from the proband were collected and whole exome sequencing was performed. Illumina Dragen and Emedgene software was used to analyze the data at Baylor Genetics. The variants were further intepreted according to ACMG guidelines and the patient's phenotype. RESULTS: Through whole-exome sequencing (WES) under the Community Texome project, he was found to have a heterozygous de novo pathogenic variant in the ATP1A3 gene located on chromosome 19q13. CONCLUSION: In retrospect, his symptomatology matches the known medical conditions associated with the ATP1A3 gene namely Alternating Hemiplegia of Childhood 2 (AHC), a rare autosomal dominant disorder with an incidence of 1 in one million. His single nucleotide variant, (c.2401G>A, p.D801N), is predicted to be damaging. The specific amino acid change p.D801N has been previously reported in ClinVar along with the allelic variant p.D801Y and both are considered pathogenic. The identification of this variant altered medical management for this patient as he was started on a calcium antagonist and has reported no further hemiplegic episodes. This case illustrates the value of implementing genomic medicine for precision therapy in underserved populations.

Cell-based Noninvasive Prenatal Testing (cbNIPT)-A Review on the Current Developments and Future Prospects.

Considering the diagnostic limitations of cfDNA-based noninvasive prenatal testing (NIPT), scientists have long been interested in isolating and analyzing rare intact fetal and trophoblast cells from maternal blood or endocervical samples to diagnose fetal genetic conditions. These cells may be scarce and difficult to isolate, but they are a direct source of pure fetal genetic material. In this review, we summarize the history of cell-based NIPT, present an updated review on its current developments, evaluate its genetic diagnostic potential, and discuss its future prospects for clinical use.

Evaluation of an automated genome interpretation model for rare disease routinely used in a clinical genetic laboratory.

PURPOSE: The analysis of exome and genome sequencing data for the diagnosis of rare diseases is challenging and time-consuming. In this study, we evaluated an artificial intelligence model, based on machine learning for automating variant prioritization for diagnosing rare genetic diseases in the Baylor Genetics clinical laboratory. METHODS: The automated analysis model was developed using a supervised learning approach based on thousands of manually curated variants. The model was evaluated on 2 cohorts. The model accuracy was determined using a retrospective cohort comprising 180 randomly selected exome cases (57 singletons, 123 trios); all of which were previously diagnosed and solved through manual interpretation. Diagnostic yield with the modified workflow was estimated using a prospective "production" cohort of 334 consecutive clinical cases. RESULTS: The model accurately pinpointed all manually reported variants as candidates. The reported variants were ranked in top 10 candidate variants in 98.4% (121/123) of trio cases, in 93.0% (53/57) of single proband cases, and 96.7% (174/180) of all cases. The accuracy of the model was reduced in some cases because of incomplete variant calling (eg, copy number variants) or incomplete phenotypic description. CONCLUSION: The automated model for case analysis assists clinical genetic laboratories in prioritizing candidate variants effectively. The use of such technology may facilitate the interpretation of genomic data for a large number of patients in the era of precision medicine.

Circulating trophoblast numbers as a potential marker for pregnancy complications.

OBJECTIVE: To explore the potential of circulating trophoblasts (TBs) as a non-invasive tool to assess placental health and predict obstetric complications. METHODS: We retrospectively reviewed maternal characteristics and pregnancy outcomes of 369 women who enrolled in our original cell-based NIPT (cbNIPT) study. The number of circulating TBs recovered from the maternal blood samples was recorded and expressed as fetal cell concentration (FCC). We evaluated if FCC can be used to predict pregnancy outcomes such as hypertensive disorders of pregnancy (HDP), fetal growth restriction, placental abruption, preterm labor, and pregnancy loss. RESULTS: Receiver operating characteristic (ROC) analysis was performed to find the best cut off value to classify FCC into a low and high FCC group, and this cut-off point was calculated as 11.1 cells per 100 ml of blood. The adjusted odds ratio (aOR) for the composite morbidity was significantly increased for the high FCC group at an aOR of 1.6. CONCLUSION: Circulating TB have the potential of predicting obstetrical complications such as HDP. Future studies, with larger sample sizes, should focus on the study of these cells as a biomarker for placental health and a possible screening or diagnostic tool for fetal genetic conditions.

Emerging technologies for prenatal diagnosis: The application of whole genome and RNA sequencing.

DNA sequencing technologies for clinical genetic testing have been rapidly evolving in recent years, and steadily become more important within the field of prenatal diagnostics. This review aims to give an overview of recent developments and to describe how they have the potential to fill the gaps of the currently clinically implemented methods for prenatal diagnosis of various genetic disorders. It has been shown for postnatal testing that whole genome sequencing provides a set of added benefits compared to exome sequencing, and it is to be expected that this will be the case for prenatal testing as well. RNA-sequencing, already used postnatally, can provide valuable complementary data to DNA-based testing, and aid in variant interpretation. While not ready for clinical implementation, emerging technologies such as long-read and Hi-C sequencing analyses might add to the toolbox for interpreting the expanding genetic data sets generated by genome-wide sequencing. Lastly, we also discuss some more practical implications of introducing these emerging technologies, which generate larger and larger genomic data sets, in the prenatal field.

De novo variants in H3-3A and H3-3B are associated with neurodevelopmental delay, dysmorphic features, and structural brain abnormalities.

The histone H3 variant H3.3, encoded by two genes H3-3A and H3-3B, can replace canonical isoforms H3.1 and H3.2. H3.3 is important in chromatin compaction, early embryonic development, and lineage commitment. The role of H3.3 in somatic cancers has been studied extensively, but its association with a congenital disorder has emerged just recently. Here we report eleven de novo missense variants and one de novo stop-loss variant in H3-3A (n = 6) and H3-3B (n = 6) from Baylor Genetics exome cohort (n = 11) and Matchmaker Exchange (n = 1), of which detailed phenotyping was conducted for 10 individuals (H3-3A = 4 and H3-3B = 6) that showed major phenotypes including global developmental delay, short stature, failure to thrive, dysmorphic facial features, structural brain abnormalities, hypotonia, and visual impairment. Three variant constructs (p.R129H, p.M121I, and p.I52N) showed significant decrease in protein expression, while one variant (p.R41C) accumulated at greater levels than wild-type control. One H3.3 variant construct (p.R129H) was found to have stronger interaction with the chaperone death domain-associated protein 6.

Overview and recent developments in cell-based noninvasive prenatal testing.

Investigators have long been interested in the natural phenomenon of fetal and placental cell trafficking into the maternal circulation. The scarcity of these circulating cells makes their detection and isolation technically challenging. However, as a DNA source of fetal origin not mixed with maternal DNA, they have the potential of considerable benefit over circulating cell-free DNA-based noninvasive prenatal genetic testing (NIPT). Endocervical trophoblasts, which are less rare but more challenging to recover are also being investigated as an approach for cell-based NIPT. We review published studies from around the world describing both forms of cell-based NIPT and highlight the different approaches' advantages and drawbacks. We also offer guidance for developing a sound cell-based NIPT protocol.

Use of amplicon-based sequencing for testing fetal identity and monogenic traits with Single Circulating Trophoblast (SCT) as one form of cell-based NIPT.

A major challenge for cell-based non-invasive prenatal testing (NIPT) is to distinguish individual presumptive fetal cells from maternal cells in female pregnancies. We have sought a rapid, robust, versatile, and low-cost next-generation sequencing method to facilitate this process. Toward this goal, single isolated cells underwent whole genome amplification prior to genotyping. Multiple highly polymorphic genomic regions (including HLA-A and HLA-B) with 10-20 very informative single nucleotide polymorphisms (SNPs) within a 200 bp interval were amplified with a modified method based on other publications. To enhance the power of cell identification, approximately 40 Human Identification SNP (Applied Biosystems) test amplicons were also utilized. Using SNP results to compare to sex chromosome data from NGS as a reliable standard, the true positive rate for genotyping was 83.4%, true negative 6.6%, false positive 3.3%, and false negative 6.6%. These results would not be sufficient for clinical diagnosis, but they demonstrate the general validity of the approach and suggest that deeper genotyping of single cells could be completely reliable. A paternal DNA sample is not required using this method. The assay also successfully detected pathogenic variants causing Tay Sachs disease, cystic fibrosis, and hemoglobinopathies in single lymphoblastoid cells, and disease-causing variants in three cell-based NIPT cases. This method could be applicable for any monogenic diagnosis.

The effect of maternal body mass index and gestational age on circulating trophoblast yield in cell-based noninvasive prenatal testing.

OBJECTIVE: To examine the effects of maternal body mass index (BMI) and gestational age (GA) on the number of single circulating trophoblasts (SCT). METHODS: Maternal blood was collected in 20 to 40 mL. All singleton pregnant women at any gestation were recruited. Trophoblasts were recovered by immunomagnetic enrichment and stained for cytokeratin and CD45. Candidate trophoblasts were identified by fluorescence microscopy. RESULTS: Blood samples were collected from 425 singleton pregnancies from April 2018 to December 2019. At least one candidate cell was identified in 88% (373/425). There was an inverse correlation between trophoblasts yield and increasing BMI (r = -0.19, P < .001). The mean ± SD number of trophoblasts/mL was 0.12 ± 0.22 in the underweight group (n = 5), 0.23 ± 0.25 in the normal weight (n = 169), 0.18 ± 0.19 in the overweight (n = 114), and 0.13 ± 0.15 in the obese (n = 109). Significantly more cells were identified in the normal weight than those in the obese (P = .001). In addition, the mean ± SD number of cells/mL was 0.21 ± 0.21 at GA of 10 to 14 weeks (n = 260), 0.14 ± 0.23 at GA ≥15 (n = 102) and 0.12 ± 0.12 at GA <10 (n = 63); P < .001. CONCLUSION: The lower number of SCT was identified from the samples of women with a high BMI. Cell recovery for SCT testing seems optimal at GA of 10 to 14 weeks, but earlier and later testing is still possible.

Validation Studies for Single Circulating Trophoblast Genetic Testing as a Form of Noninvasive Prenatal Diagnosis.

It has long been appreciated that genetic analysis of fetal or trophoblast cells in maternal blood could revolutionize prenatal diagnosis. We implemented a protocol for single circulating trophoblast (SCT) testing using positive selection by magnetic-activated cell sorting and single-cell low-coverage whole-genome sequencing to detect fetal aneuploidies and copy-number variants (CNVs) at ∼1 Mb resolution. In 95 validation cases, we identified on average 0.20 putative trophoblasts/mL, of which 55% were of high quality and scorable for both aneuploidy and CNVs. We emphasize the importance of analyzing individual cells because some cells are apoptotic, in S-phase, or otherwise of poor quality. When two or more high-quality trophoblast cells were available for singleton pregnancies, there was complete concordance between all trophoblasts unless there was evidence of confined placental mosaicism. SCT results were highly concordant with available clinical data from chorionic villus sampling (CVS) or amniocentesis procedures. Although determining the exact sensitivity and specificity will require more data, this study further supports the potential for SCT testing to become a diagnostic prenatal test.

Reliable detection of subchromosomal deletions and duplications using cell-based noninvasive prenatal testing.

OBJECTIVE: To gather additional data on the ability to detect subchromosomal abnormalities of various sizes in single fetal cells isolated from maternal blood, using low-coverage shotgun next-generation sequencing for cell-based noninvasive prenatal testing (NIPT). METHOD: Fetal trophoblasts were recovered from approximately 30 mL of maternal blood using maternal white blood cell depletion, density-based cell separation, immunofluorescence staining, and high-resolution scanning. These trophoblastic cells were picked as single cells and underwent whole genome amplification for subsequent genome-wide copy number analysis and genotyping to confirm the fetal origin of the cells. RESULTS: Applying our fetal cell isolation method to a series of 125 maternal blood samples, we detected on average 4.17 putative fetal cells/sample. The series included 15 cases with clinically diagnosed fetal aneuploidies and five cases with subchromosomal abnormalities. This method was capable of detecting findings that were 1 to 2 Mb in size, and all were concordant with the microarray or karyotype data obtained on a fetal sample. A minority of fetal cells showed evidence of genome degradation likely related to apoptosis. CONCLUSION: We demonstrate that this cell-based NIPT method has the capacity to reliably diagnose fetal chromosomal abnormalities down to 1 to 2 Mb in size.

Comparison of fractionation proteomics for local SWATH library building.

For data-independent acquisition by means of sequential window acquisition of all theoretical fragment ion spectra (SWATH), a reference library of data-dependent acquisition (DDA) runs is typically used to correlate the quantitative data from the fragment ion spectra with peptide identifications. The quality and coverage of such a reference library is therefore essential when processing SWATH data. In general, library sizes can be increased by reducing the impact of DDA precursor selection with replicate runs or fractionation. However, these strategies can affect the match between the library and SWATH measurement, and thus larger library sizes do not necessarily correspond to improved SWATH quantification. Here, three fractionation strategies to increase local library size were compared to standard library building using replicate DDA injection: protein SDS-PAGE fractionation, peptide high-pH RP-HPLC fractionation and MS-acquisition gas phase fractionation. The impact of these libraries on SWATH performance was evaluated in terms of the number of extracted peptides and proteins, the match quality of the peptides and the extraction reproducibility of the transitions. These analyses were conducted using the hydrophilic proteome of differentiating human embryonic stem cells. Our results show that SWATH quantitative results and interpretations are affected by choice of fractionation technique. Data are available via ProteomeXchange with identifier PXD006190.

Extracting histones for the specific purpose of label-free MS.

Extracting histones from cells is the first step in studies that aim to characterize histones and their post-translational modifications (hPTMs) with MS. In the last decade, label-free quantification is more frequently being used for MS-based histone characterization. However, many histone extraction protocols were not specifically designed for label-free MS. While label-free quantification has its advantages, it is also very susceptible to technical variation. Here, we adjust an established histone extraction protocol according to general label-free MS guidelines with a specific focus on minimizing sample handling. These protocols are first evaluated using SDS-PAGE. Hereafter, a selection of extraction protocols was used in a complete histone workflow for label-free MS. All protocols display nearly identical relative quantification of hPTMs. We thus show that, depending on the cell type under investigation and at the cost of some additional contaminating proteins, minimizing sample handling can be done during histone isolation. This allows analyzing bigger sample batches, leads to reduced technical variation and minimizes the chance of in vitro alterations to the hPTM snapshot. Overall, these results allow researchers to determine the best protocol depending on the resources and goal of their specific study. Data are available via ProteomeXchange with identifier PXD002885.

Evidence for feasibility of fetal trophoblastic cell-based noninvasive prenatal testing.

OBJECTIVE: The goal was to develop methods for detection of chromosomal and subchromosomal abnormalities in fetal cells in the mother's circulation at 10-16 weeks' gestation using analysis by array comparative genomic hybridization (CGH) and/or next-generation sequencing (NGS). METHOD: Nucleated cells from 30 mL of blood collected at 10-16 weeks' gestation were separated from red cells by density fractionation and then immunostained to identify cytokeratin positive and CD45 negative trophoblasts. Individual cells were picked and subjected to whole genome amplification, genotyping, and analysis by array CGH and NGS. RESULTS: Fetal cells were recovered from most samples as documented by Y chromosome PCR, short tandem repeat analysis, array CGH, and NGS including over 30 normal male cells, one 47,XXY cell from an affected fetus, one trisomy 18 cell from an affected fetus, nine cells from a trisomy 21 case, three normal cells and one trisomy 13 cell from a case with confined placental mosaicism, and two chromosome 15 deletion cells from a case known by CVS to have a 2.7 Mb de novo deletion. CONCLUSION: We believe that this is the first report of using array CGH and NGS whole genome sequencing to detect chromosomal abnormalities in fetal trophoblastic cells from maternal blood. © 2016 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.

Assessing the impact of minimizing arginine conversion in fully defined SILAC culture medium in human embryonic stem cells.

We present a fully defined culture system (adapted Essential8TM [E8TM ] medium in combination with vitronectin) for human embryonic stem cells that can be used for SILAC purposes. Although a complete incorporation of the labels was observed after 4 days in culture, over 90% of precursors showed at least 10% conversion. To reduce this arginine conversion, E8TM medium was modified by adding (1) l-proline, (2) l-ornithine, (3) Nω -hydroxy-nor-l-arginine acetate, or by (4) lowering the arginine concentration. Reduction of arginine conversion was best obtained by adding 5 mM l-ornithine, followed by 3.5 mM l-proline and by lowering the arginine concentration in the medium to 99.5 µM. No major changes in pluripotency and cell amount could be observed for the adapted E8TM media with ornithine and proline. However, our subsequent ion mobility assisted data-independent acquisition (high-definition MS) proteome analysis cautions for ongoing changes in the proteome when aiming at longer term suppression of arginine conversion.

Histone proteolysis: a proposal for categorization into 'clipping' and 'degradation'.

We propose for the first time to divide histone proteolysis into "histone degradation" and the epigenetically connoted "histone clipping". Our initial observation is that these two different classes are very hard to distinguish both experimentally and biologically, because they can both be mediated by the same enzymes. Since the first report decades ago, proteolysis has been found in a broad spectrum of eukaryotic organisms. However, the authors often not clearly distinguish or determine whether degradation or clipping was studied. Given the importance of histone modifications in epigenetic regulation we further elaborate on the different ways in which histone proteolysis could play a role in epigenetics. Finally, unanticipated histone proteolysis has probably left a mark on many studies of histones in the past. In conclusion, we emphasize the significance of reviving the study of histone proteolysis both from a biological and an experimental perspective. Also watch the Video Abstract.