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.

Determining the characteristics of genetic disorders that predict inclusion in newborn genomic sequencing programs.

Over 30 international research studies and commercial laboratories are exploring the use of genomic sequencing to screen apparently healthy newborns for genetic disorders. These programs have individualized processes for determining which genes and genetic disorders are queried and reported in newborns. We compared lists of genes from 26 research and commercial newborn screening programs and found substantial heterogeneity among the genes included. A total of 1,750 genes were included in at least one newborn genome sequencing program, but only 74 genes were included on >80% of gene lists, 16 of which are not associated with conditions on the Recommended Uniform Screening Panel. We used a linear regression model to explore factors related to the inclusion of individual genes across programs, finding that a high evidence base as well as treatment efficacy were two of the most important factors for inclusion. We applied a machine learning model to predict how suitable a gene is for newborn sequencing. As knowledge about and treatments for genetic disorders expand, this model provides a dynamic tool to reassess genes for newborn screening implementation. This study highlights the complex landscape of gene list curation among genomic newborn screening programs and proposes an empirical path forward for determining the genes and disorders of highest priority for newborn screening programs.

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.

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.

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.

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.

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.