Long-read sequencing and optical genome mapping identify causative gene disruptions in noncoding sequence in two patients with neurologic disease and known chromosome abnormalities.

Despite advances in next generation sequencing (NGS), genetic diagnoses remain elusive for many patients with neurologic syndromes. Long-read sequencing (LRS) and optical genome mapping (OGM) technologies improve upon existing capabilities in the detection and interpretation of structural variation in repetitive DNA, on a single haplotype, while also providing enhanced breakpoint resolution. We performed LRS and OGM on two patients with known chromosomal rearrangements and inconclusive Sanger or NGS. The first patient, who had epilepsy and developmental delay, had a complex translocation between two chromosomes that included insertion and inversion events. The second patient, who had a movement disorder, had an inversion on a single chromosome disrupted by multiple smaller inversions and insertions. Sequence level resolution of the rearrangements identified pathogenic breaks in noncoding sequence in or near known disease-causing genes with relevant neurologic phenotypes (MBD5, NKX2-1). These specific variants have not been reported previously, but expected molecular consequences are consistent with previously reported cases. As the use of LRS and OGM technologies for clinical testing increases and data analyses become more standardized, these methods along with multiomic data to validate noncoding variation effects will improve diagnostic yield and increase the proportion of probands with detectable pathogenic variants for known genes implicated in neurogenetic disease.

Data-driven prioritization of genetic disorders for global genomic newborn screening programs.

Genomic sequencing is poised to expand newborn screening for treatable childhood-onset disorders. Over 30 international research studies and companies are exploring its use, collectively aiming to screen more than 500,000 infants. A key challenge is determining which genes to include in screening. Among 27 newborn sequencing programs, the number of genes analyzed ranged from 134 to 4,299, with only 74 genes included by over 80% of programs. To understand this variability, we assembled a dataset with 25 characteristics of 4,389 genes included in any program and used a multivariate regression analysis to identify characteristics associated with inclusion across programs. These characteristics included presence on the US Recommended Uniform Screening panel, evidence regarding the natural history of disease, and efficacy of treatment. We then used a machine learning model to generate a ranked list of genes, offering a data-driven approach to the future prioritization of disorders for public health newborn screening efforts.

Lymphangioleiomyomatosis Association with Underlying Genotype in Patients with Tuberous Sclerosis Complex.

Rationale: Lymphangioleiomyomatosis (LAM) is a female-predominant lung disease caused by mutations in the tuberous sclerosis complex (TSC) genes TSC1 and TSC2.Objectives: To examine the association between TSC mutation subtypes and the prevalence of LAM in women with TSC.Methods: Adult women seen at the Cincinnati Children's Hospital Medical Center's TSC clinic were stratified into the following three groups: those with TSC1 mutation, those with TSC2 mutation, and those with no mutation identified (NMI). Individual TSC manifestations were ascertained by blinded review of chest computed tomographic scans (LAM, multifocal micronodular pneumocyte hyperplasia, and sclerotic bone lesions) and chart review (all other manifestations). The association between mutation status and TSC manifestations was assessed by the Wilcoxon rank-sum test.Results: Our cohort consisted of 55 TSC women, including 30/55 (55%) with TSC2, 12/55 (22%) with TSC1, and 13/55 (23%) with NMI. Twenty-three women (42%) had characteristic cysts consistent with LAM, of whom 16 had TSC2 mutations and seven had NMI. The prevalence of LAM was higher in women with TSC2 mutations compared with women with TSC1 mutations (16/29 [55%] vs. 0/12; P = 0.003). Similarly, renal angiomyolipomas were more common in women with TSC2 mutations compared with women with TSC1 mutations (29/30 [97%] vs. 6/12 [50%]; P = 0.01). There was no association between TSC mutation subtype and the presence of multifocal micronodular pneumocyte hyperplasia, sclerotic bone lesions, and skin or brain involvement. Serum VEGF-D (vascular endothelial growth factor-D) concentrations (median [95% confidence interval]) tended to be higher in patients harboring TSC2 mutations compared with patients with TSC1 mutations (725 pg/ml [612-1,317] vs. 331 pg/ml [284-406]; P = 0.03) and in patients with LAM compared with patients without LAM (725 pg/ml [563-1,609] vs. 429 pg/ml [357-773]; P = 0.02).Conclusions: LAM and angiomyolipomas are more common in women with TSC harboring TSC2 mutations compared with women with TSC1 mutations. Serum VEGF-D is a useful biomarker to suggest the presence of LAM in women with TSC.

Confirmatory testing illustrates additional risks for structural sex chromosome abnormalities in fetuses with a non-invasive prenatal screen positive for monosomy X.

More and more women rely on non-invasive prenatal screening (NIPS) to detect fetal sex and risk for aneuploidy. The testing applies massively parallel sequencing or single nucleotide polymorphism (SNP) microarray to circulating cell-free DNA to determine relative copy number. In addition to trisomies 13, 18, and 21, some labs offer screening for sex chromosome abnormalities as part of their test. In this study, an index neonate screened positive for monosomy X and had discordant postnatal chromosomes indicating an X;autosome translocation. This patient prompted a retrospective chart review for similar cases at a large NIPS testing center. The review found 28 patients with an abnormal NIPS for monosomy X who were eventually diagnosed with additional discrepant structural sex chromosome abnormalities including translocations, isochromosomes, deletions, rings, markers, and uniparental disomy. The majority of these were mosaic with monosomy X, but in seven cases, there was no evidence of mosaicism on confirmatory testing. The identification of multiple sex chromosome aneuploidies in these cases supports the need for additional genetic counseling prior to NIPS testing and following abnormal NIPS results that are positive for monosomy X. This finding broadens our knowledge about the variable outcomes of positive monosomy X NIPS results and emphasizes the importance of confirmatory testing and clinical follow up for these patients.

The Genomics Research and Innovation Network: creating an interoperable, federated, genomics learning system.

PURPOSE: Clinicians and researchers must contextualize a patient's genetic variants against population-based references with detailed phenotyping. We sought to establish globally scalable technology, policy, and procedures for sharing biosamples and associated genomic and phenotypic data on broadly consented cohorts, across sites of care. METHODS: Three of the nation's leading children's hospitals launched the Genomic Research and Innovation Network (GRIN), with federated information technology infrastructure, harmonized biobanking protocols, and material transfer agreements. Pilot studies in epilepsy and short stature were completed to design and test the collaboration model. RESULTS: Harmonized, broadly consented institutional review board (IRB) protocols were approved and used for biobank enrollment, creating ever-expanding, compatible biobanks. An open source federated query infrastructure was established over genotype-phenotype databases at the three hospitals. Investigators securely access the GRIN platform for prep to research queries, receiving aggregate counts of patients with particular phenotypes or genotypes in each biobank. With proper approvals, de-identified data is exported to a shared analytic workspace. Investigators at all sites enthusiastically collaborated on the pilot studies, resulting in multiple publications. Investigators have also begun to successfully utilize the infrastructure for grant applications. CONCLUSIONS: The GRIN collaboration establishes the technology, policy, and procedures for a scalable genomic research network.

Correction: The Genomics Research and Innovation Network: creating an interoperable, federated, genomics learning system.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Homozygous missense variant in BMPR1A resulting in BMPR signaling disruption and syndromic features.

BACKGROUND: The bone morphogenetic protein (BMP) pathway is known to play an imperative role in bone, cartilage, and cardiac tissue formation. Truncating, heterozygous variants, and deletions of one of the essential receptors in this pathway, Bone Morphogenetic Protein Receptor Type1A (BMPR1A), have been associated with autosomal dominant juvenile polyposis. Heterozygous deletions have also been associated with cardiac and minor skeletal anomalies. Populations with atrioventricular septal defects are enriched for rare missense BMPR1A variants. METHODS: We report on a patient with a homozygous missense variant in BMPR1A causing skeletal abnormalities, growth failure a large atrial septal defect, severe subglottic stenosis, laryngomalacia, facial dysmorphisms, and developmental delays. RESULTS: Functional analysis of this variant shows increased chondrocyte death for cells with the mutated receptor, increased phosphorylated R-Smads1/5/8, and loss of Sox9 expression mediated by decreased phosphorylation of p38. CONCLUSION: This homozygous missense variant in BMPR1A appears to cause a distinct clinical phenotype.

Using human sequencing to guide craniofacial research.

A recent convergence of technological innovations has re-energized the ability to apply genetics to research in human craniofacial development. Next-generation exome and whole genome sequencing have significantly dropped in price, making it relatively trivial to sequence and analyze patients and families with congenital craniofacial anomalies. A concurrent revolution in genome editing with the use of the CRISPR-Cas9 system enables the rapid generation of animal models, including mouse, which can precisely recapitulate human variants. Here, we summarize the choices currently available to the research community. We illustrate this approach with the study of a family with a novel craniofacial syndrome with dominant inheritance pattern. The genomic analysis suggested a causal variant in AMOTL1 which we modeled in mice. We also made a novel deletion allele of Amotl1. Our results indicate that Amotl1 is not required in the mouse for survival to weaning. Mice carrying the variant identified in the human sequencing studies, however, do not survive to weaning in normal ratios. The cause of death is not understood for these mice complicating our conclusions about the pathogenicity in the index patient. Thus, we highlight some of the powerful opportunities and confounding factors confronting current craniofacial genetic research.

Copb2 is essential for embryogenesis and hypomorphic mutations cause human microcephaly.

Primary microcephaly is a congenital brain malformation characterized by a head circumference less than three standard deviations below the mean for age and sex and results in moderate to severe mental deficiencies and decreased lifespan. We recently studied two children with primary microcephaly in an otherwise unaffected family. Exome sequencing identified an autosomal recessive mutation leading to an amino acid substitution in a WD40 domain of the highly conserved Coatomer Protein Complex, Subunit Beta 2 (COPB2). To study the role of Copb2 in neural development, we utilized genome-editing technology to generate an allelic series in the mouse. Two independent null alleles revealed that Copb2 is essential for early stages of embryogenesis. Mice homozygous for the patient variant (Copb2R254C/R254C) appear to have a grossly normal phenotype, likely due to differences in corticogenesis between the two species. Strikingly, mice heterozygous for the patient mutation and a null allele (Copb2R254C/Zfn) show a severe perinatal phenotype including low neonatal weight, significantly increased apoptosis in the brain, and death within the first week of life. Immunostaining of the Copb2R254C/Zfnbrain revealed a reduction in layer V (CTIP2+) neurons, while the overall cell density of the cortex is unchanged. Moreover, neurospheres derived from animals with Copb2 variants grew less than control. These results identify a general requirement for COPB2 in embryogenesis and a specific role in corticogenesis. We further demonstrate the utility of CRISPR-Cas9 generated mouse models in the study of potential pathogenicity of variants of potential clinical interest.

Mutations in SLC25A46, encoding a UGO1-like protein, cause an optic atrophy spectrum disorder.

Dominant optic atrophy (DOA) and axonal peripheral neuropathy (Charcot-Marie-Tooth type 2, or CMT2) are hereditary neurodegenerative disorders most commonly caused by mutations in the canonical mitochondrial fusion genes OPA1 and MFN2, respectively. In yeast, homologs of OPA1 (Mgm1) and MFN2 (Fzo1) work in concert with Ugo1, for which no human equivalent has been identified thus far. By whole-exome sequencing of patients with optic atrophy and CMT2, we identified four families with recessive mutations in SLC25A46. We demonstrate that SLC25A46, like Ugo1, is a modified carrier protein that has been recruited to the outer mitochondrial membrane and interacts with the inner membrane remodeling protein mitofilin (Fcj1). Loss of function in cultured cells and in zebrafish unexpectedly leads to increased mitochondrial connectivity, while severely affecting the development and maintenance of neurons in the fish. The discovery of SLC25A46 strengthens the genetic overlap between optic atrophy and CMT2 while exemplifying a new class of modified solute transporters linked to mitochondrial dynamics.

Acrofacial Dysostosis, Cincinnati Type, a Mandibulofacial Dysostosis Syndrome with Limb Anomalies, Is Caused by POLR1A Dysfunction.

We report three individuals with a cranioskeletal malformation syndrome that we define as acrofacial dysostosis, Cincinnati type. Each individual has a heterozygous mutation in POLR1A, which encodes a core component of RNA polymerase 1. All three individuals exhibit varying degrees of mandibulofacial dysostosis, and two additionally have limb anomalies. Consistent with this observation, we discovered that polr1a mutant zebrafish exhibited cranioskeletal anomalies mimicking the human phenotype. polr1a loss of function led to perturbed ribosome biogenesis and p53-dependent cell death, resulting in a deficiency of neural-crest-derived skeletal precursor cells and consequently craniofacial anomalies. Our findings expand the genotypic and phenotypic heterogeneity of congenital acrofacial disorders caused by disruption of ribosome biogenesis.

A mutation in FRIZZLED2 impairs Wnt signaling and causes autosomal dominant omodysplasia.

Autosomal dominant omodysplasia is a rare skeletal dysplasia characterized by short humeri, radial head dislocation, short first metacarpals, facial dysmorphism and genitourinary anomalies. We performed next-generation whole-exome sequencing and comparative analysis of a proband with omodysplasia, her unaffected parents and her affected daughter. We identified a de novo mutation in FRIZZLED2 (FZD2) in the proband and her daughter that was not found in unaffected family members. The FZD2 mutation (c.1644G>A) changes a tryptophan residue at amino acid 548 to a premature stop (p.Trp548*). This altered protein is still produced in vitro, but we show reduced ability of this mutant form of FZD2 to interact with its downstream target DISHEVELLED. Furthermore, expressing the mutant form of FZD2 in vitro is not able to facilitate the cellular response to canonical Wnt signaling like wild-type FZD2. We therefore conclude that the FRIZZLED2 mutation is a de novo, novel cause for autosomal dominant omodysplasia.

Detection and reporting of homozygosity associated with consanguinity in the clinical laboratory.

The detection of consanguinity by the presence of multiple regions of homozygosity (ROH) is not an uncommon occurrence in clinical laboratories performing SNP microarray analysis. Reporting practices amongst laboratories are highly variable, due in part to differences in testing platforms, threshold parameters, language utilized, and laboratory policies. While guidance documents have provided a framework for detection and reporting practices, and will doubtless serve to harmonize the field, there are still many facets of the testing that remain at the discretion of the performing laboratory. Clinician and patient education remain a high priority. In the clinical laboratory, these homozygous segments are often examined to identify genes associated with a phenotype that matches that of the proband and autosomal recessive inheritance. While the detection of these ROH is possible with whole genome sequencing, it currently requires special algorithms be utilized, an uncommon practice in most clinical laboratories currently performing this type of testing.

Keutel syndrome: report of two novel MGP mutations and discussion of clinical overlap with arylsulfatase E deficiency and relapsing polychondritis.

Keutel syndrome is a rare, autosomal recessive disorder characterized by diffuse cartilage calcification, peripheral pulmonary artery stenosis, midface retrusion, and short distal phalanges. To date, 28 patients from 18 families have been reported, and five mutations in the matrix Gla protein gene (MGP) have been identified. The matrix Gla protein (MGP) is a vitamin K-dependent extracellular protein that functions as a calcification inhibitor through incompletely understood mechanisms. We present the clinical manifestations of three affected siblings from a consanguineous Turkish family, in whom we detected the sixth MGP mutation (c.79G>T, which predicts p.E27X) and a fourth unrelated patient in whom we detected the seventh MGP mutation, a partial deletion of exon 4. Both mutations predict complete loss of MGP function. One of the patients presented initially with a working diagnosis of relapsing polychondritis. Clinical features suggestive of Keutel syndrome were also observed in one additional unrelated patient who was later found to have a deletion of arylsulfatase E, consistent with a diagnosis of X-linked recessive chondrodysplasia punctata. Through a discussion of these cases, we highlight the clinical overlap of Keutel syndrome, X-linked chondrodysplasia punctata, and the inflammatory disease relapsing polychondritis.

Variable approaches to genetic counseling for microarray regions of homozygosity associated with parental relatedness.

SNP microarrays are capable of detecting regions of homozygosity (ROH) which can suggest parental relatedness. This study was designed to describe pre- and post-test counseling practices of genetics professionals regarding ROH, explore perceived comfort and ethical concerns in the follow-up of such results, demonstrate awareness of laws surrounding duty to report consanguinity and incest, and allow respondents to share their personal experiences with results suggesting a parental relationship. A 35 question survey was administered to 240 genetic counselors and geneticists who had ordered or counseled for SNP microarray. The results are presented using descriptive statistics. There was variation in both pre- and post-test counseling practices of genetics professionals. Twenty-five percent of respondents reported pre-test counseling that ROH can indicate parental relatedness. The most commonly reported ethical concern was disclosure of findings suggesting parental relatedness to parents of the patient; only 48.4% reported disclosing parental relatedness when indicated. Fifty-seven percent felt comfortable receiving results suggesting parental consanguinity while 17% felt comfortable receiving results suggesting parental incest. Twenty percent of respondents were extremely/moderately familiar with the laws about duty to report incest. Personal experiences in post-test counseling included both parental acknowledgement and denial of relatedness. This study highlights the differences in genetics professionals' pre- and post-test counseling practices, comfort, and experiences surrounding parental relatedness suggested by SNP microarray results. It identifies a need for professional organizations to offer guidance to genetics professionals about how to respond to and counsel for molecular results suggesting parental consanguinity or incest.

Genetic testing practices in infants with congenital heart disease.

OBJECTIVE: Clinical genetic testing is expanding rapidly, but the application of new testing has not been reported in an unselected, comprehensive congenital heart disease (CHD) patient population. This study aims to identify cytogenetic testing practices and diagnostic yield in infants with CHD as an important first step toward understanding clinical utility of dedicated cytogenetic testing. We hypothesized that chromosome microarray analysis (CMA) would identify genetic abnormalities underlying both syndromic and isolated CHD. DESIGN: This is a single institution retrospective study that characterizes cytogenetic testing practices and diagnostic yield for all cytogenetic testing in each infant identified with CHD over a 32-month period. CHD was classified by type, complexity, and presence or absence of extracardiac anomalies. RESULTS: Among the 1087 infants identified with CHD by echocardiogram, 277 infants (25%) had some form of cytogenetic testing, including karyotype, fluorescence in situ hybridization, and/or CMA. Forty-one percent of infants who had cytogenetic testing had more than one test. CMA was performed in 121 patients (11%), and abnormalities (both clinically significant and variants of unknown significance) were identified in 35/121 (29%). Forty-nine percent of CMA abnormalities were in patients with apparently isolated nonsyndromic CHD. CONCLUSIONS: This single institution study identified that only 25% of infants with CHD underwent cytogenetic testing, indicating possible underutilization of testing in this age group. The high multiple testing rate indicates a need for improved guidelines for cost effective testing approaches. The diagnostic yield in this study suggests that CMA is a particularly useful first screening test when a specific syndrome is not clinically identifiable. Larger studies investigating cardiac lesion-specific diagnostic yield in isolated CHD are warranted.

Regions of homozygosity identified by SNP microarray analysis aid in the diagnosis of autosomal recessive disease and incidentally detect parental blood relationships.

PURPOSE: The purpose of this study was to document the ability of single-nucleotide polymorphism microarray to identify copy-neutral regions of homozygosity, demonstrate clinical utility of regions of homozygosity, and discuss ethical/legal implications when regions of homozygosity are associated with a parental blood relationship. METHODS: Study data were compiled from consecutive samples sent to our clinical laboratory over a 3-year period. A cytogenetics database identified patients with at least two regions of homozygosity >10 Mb on two separate chromosomes. A chart review was conducted on patients who met the criteria. RESULTS: Of 3,217 single-nucleotide polymorphism microarrays, 59 (1.8%) patients met inclusion criteria. The percentage of homozygosity ranged from 0.9 to 30.1%, indicating parental relationships from distant to first-degree relatives. First-degree kinship was suspected in the parents of at least 11 patients with regions of homozygosity covering >21.3% of their autosome. In four patients from two families, homozygosity mapping discovered a candidate gene that was sequenced to identify a clinically significant mutation. CONCLUSION: This study demonstrates clinical utility in the identification of regions of homozygosity, as these regions may aid in diagnosis of the patient. This study establishes the need for careful reporting, thorough pretest counseling, and careful electronic documentation, as microarray has the capability of detecting previously unknown/unreported relationships.

Variability in laboratory reporting practices for regions of homozygosity indicating parental relatedness as identified by SNP microarray testing.

PURPOSE: Single-nucleotide polymorphism (SNP) microarrays are capable of detecting regions of homozygosity (ROH) that can suggest parental consanguinity or incest. This study was designed to describe the variable reporting practices of clinical laboratories in the United States regarding ROH found on SNP microarray tests, to discuss the follow-up practices of laboratory personnel when findings of ROH indicate consanguinity or incest, and to highlight the legal and ethical dilemmas faced by workers who have discovered these incidental findings. METHODS: A 20-question survey was administered to microarray experts at 18 laboratories offering clinical SNP microarray tests. The results are presented using descriptive statistics. RESULTS: There was variability in laboratory SNP microarray reporting practices with respect to information and interpretation of ROH findings. All the laboratories agreed that they have a duty to inform the ordering physician about results suggesting consanguinity or incest, but the follow-through practices varied among laboratories. CONCLUSIONS: This study discovered variability in reporting practices and follow-up procedures for microarray results that suggest parental consanguinity or incest. Our findings highlight the need for laboratory guidelines to standardize reporting practices for SNP microarray and other tests that are capable of detecting ROH.

Analysis of Ellis van Creveld syndrome gene products: implications for cardiovascular development and disease.

Mutations identified in a cohort of patients with atrioventricular septal defects as a part of Ellis van Creveld syndrome (EvC syndrome) led us to study the role of two non-homologous genes, EVC and LBN, in heart development and disease pathogenesis. To address the cause of locus heterogeneity resulting in an indistinguishable heart-hand phenotype, we carried out in situ hybridization and immunofluorescence and identified co-localization of Evc and Lbn mRNA and protein. In the heart, expression was identified to be strongest in the secondary heart field, including both the outflow tract and the dorsal mesenchymal protrusion, but was also found in mesenchymal structures of the atrial septum and the atrioventricular cushions. Finally, we studied the transcriptional hierarchy of EVC and LBN but did not find any evidence of direct transcriptional interregulation between the two. Due to the locus heterogeneity of human mutations predicted to result in a loss of protein function, a bidirectional genomic organization and overlapping expression patterns, we speculate that these proteins function coordinately in cardiac development and that loss of this coordinate function results in the characteristics of EvC syndrome.