Biallelic hypomorphic variants in PRORP cause the rare autosomal recessive disorder combined oxidative phosphorylation deficiency type 54 (COXPD54). COXPD54 encompasses a clinical spectrum of sensorineural hearing loss and ovarian insufficiency (Perrault syndrome) to leukodystrophy with developmental delay and epilepsy. Here, we report two new affected individuals with biallelic PRORP variants with clinical features consistent with COXPD54. One individual was homozygous for c.1505G > A p.Arg502Gln, whereas the other was compound heterozygous for c.1510C > T, p.His504Tyr and c.893C > A, p.Ser298Ter (NM_014672.4). In vitro tRNA processing assays revealed decreased mitochondrial 5′ tRNA leader cleavage by human RNase P complex with the two novel missense PRORP metallonuclease domain variants. These data provide further evidence that biallelic PRORP variants disrupt 5’ tRNA leader cleavage and are associated with a pleiotropic phenotype of COXPD54.

The CECR2 histone acetyl-lysine reader facilitates chromatin remodeling and plays a significant role in neurodevelopment. It resides within the cat eye syndrome (CES) critical region at 22q11.1q11.21. An increased copy number of this region, often as tetrasomy or a supernumerary chromosome, results in CES. The complex chromosomal arrangements and phenotypic variability have hampered the identification of the true cause(s) of CES. Patients and their clinical data were collected from multiple diagnostic and research laboratories. Exome or genome sequencing was performed in affected individuals and, when available, their relatives. Here, we describe six patients with a heterozygous single-nucleotide or small insertion/deletion variant in CECR2 and their clinical features. Variants were either loss-of-function [c.1734dupC:p.(Thr580Hisfs∗98), c.1819C>T:p.(Arg607∗), and c.2281C>T:p.(Arg761∗)] or missense in an intrinsically disordered and potentially critical region at the C terminus of the protein [c.4153C>G:p.(Gln1385Glu), c.4254C>G:p.(Phe1418Leu), and c.4283A>G:p.(Gln1428Arg)]. The main clinical features were delayed growth, relatively small head circumference, developmental delay, and speech issues/delay. Other clinical findings were variable and included intellectual disability; feeding problems; facial dysmorphism; cleft lip/palate; brain, ear, heart, or other organ abnormalities; behavioral abnormalities; and epilepsy. There was significant overlap with common CES features, including ear, heart, and brain abnormalities, intellectual disability, and growth restriction. In conclusion, we characterize a CECR2-associated neurodevelopmental disorder. The consistent molecular and clinical overlap with CES supports CECR2's role in the CES phenotype. These findings implicate diverse variant types in related neurodevelopmental disorders, underscoring CECR2's dosage sensitivity and essential function in epigenetic regulation during development.

Pathogenic variants in GNAS can cause a wide range of diseases including pseudohypoparathyroidism, pseudopseudohypoparathyroidism, McCune-Albright syndrome, among others. The specific phenotypic features that may be seen are influenced by the variant type and location in the gene, whether it causes loss or gain of function, and whether it is germline or somatic in nature. The GNAS locus is imprinted, which also results in a parent-of-origin effect. Typically, germline loss of function variants on the maternal allele are associated with variable hormonal resistances, obesity, intrauterine growth restriction, and cognitive impairment. Here, we describe a mother and daughter with a unique splicing variant near exon 5 of the GNAS gene (NM_000516.5:c.432 + 5G>A), shown to cause alternative splicing through RNA sequencing (RNA-seq), likely resulting in a loss-of-function effect. Segregation testing revealed that the variant arose de novo in the mother, and phasing showed it was on her paternal allele. The resultant phenotype includes a SHOX deficiency-like disorder with Madelung deformity in the mother, and significant growth restriction with brachydactyly in the daughter, further expanding the phenotypic spectrum of GNAS inactivation disorders.

Germline ZFX variants are associated with an X-linked neurodevelopmental disorder, with 14 males and 16 females reported to date. We describe a 20-year-old female with a heterozygous ZFX frameshift variant, p.(Met666Valfs*2), identified by genome sequencing, previously reported in an affected male. She exhibited motor and speech delays with hypotonia in early childhood, and was later diagnosed with congenital heart defects, autism spectrum disorder, mild intellectual disability, and absence seizures. She further developed sensorineural hearing loss, skin hyperpigmentation, and ophthalmoplegia. Novel phenotypic features included inferior cerebellar vermian hypoplasia, hypoplastic right vertebral artery, aberrant subclavian artery, long palpebral fissures, ophthalmoplegia, skin hyperpigmentation, and a short uvula, expanding the known clinical spectrum. Female carriers of pathogenic ZFX variants demonstrate highly variable expressivity, ranging from apparently unaffected individuals to syndromic presentations. Individuals with heterozygous missense variants often exhibit hyperparathyroidism, suggesting a genotype-phenotype correlation. Reanalysis of published RNA-sequencing data identified 15 ZFX target genes involved in neurodevelopment, suggesting a role for these genes in disease pathogenesis. These findings confirm the pathogenicity of the p.(Met666Valfs*2) variant in the proband and highlight the phenotypic heterogeneity of the disorder in females. Clinical care should include cardiac and endocrine monitoring, with endocrine testing offered to unaffected females carrying missense variants.

De novo variants in CSNK2A1 cause autosomal dominant Okur-Chung neurodevelopmental syndrome (OCNDS). OCNDS has an evolving clinical phenotype predominantly characterized by intellectual disability, global delays, dysmorphic features, and immunological manifestations. Microcephaly, defined as a small head circumference, is not widely recognized as a classical clinical presentation. Here, we describe four individuals from three unrelated families who shared several clinical features characteristic of an underlying syndromic neurodevelopmental condition. Trio clinical exome and research genome sequencing revealed that all affected individuals had heterozygous pathogenic missense variants in CSNK2A1. Two variants (c.468T>A p.Asp156Glu and c.149A>G p.Tyr50Cys) were de novo and previously reported, but the third variant (c.137G>T p.Gly46Val) is novel and segregated in two affected individuals in a family. This adds to growing evidence of inherited disease-causing variants in CSNK2A1, an observation reported only twice previously. A detailed phenotypic analysis of our cohort together with those individuals reported in the literature revealed that OCNDS individuals, on average, have a smaller head circumference with one-third presenting with microcephaly. We also show that the incidence of microcephaly is significantly correlated with the location of the variant in the encoded protein. Our findings suggest that small head circumference is a common but under-recognized feature of OCNDS, which may not be apparent at birth.

Polypyrimidine tract-binding protein 1 (PTBP1) is a heterogeneous nuclear ribonucleoprotein primarily known for its alternative splicing activity. It shuttles between the nucleus and cytoplasm via partially overlapping N-terminal nuclear localization (NLS) and export (NES) signals. Despite its fundamental role in cell growth and differentiation, its involvement in human disease remains poorly understood. We identified 27 individuals from 25 families harboring de novo or inherited pathogenic variants - predominantly start-loss (89%) and, to a lesser extent, missense (11%) - affecting NES/NLS motifs. Affected individuals presented with a syndromic neurodevelopmental disorder and variable skeletal dysplasia with disproportionate short stature with short limbs. Intellectual functioning ranged from normal to moderately delayed. Start-loss variants led to translation initiation from an alternative downstream in-frame methionine, resulting in loss of the NES and the first half of the bipartite NLS, and increased cytoplasmic stability. Start-loss and missense variants shared a DNA methylation episignature in peripheral blood and altered nucleocytoplasmic distribution in vitro and in vivo with preferential accumulation in processing bodies, causing aberrant gene expression but normal RNA splicing. Transcriptomic analysis of patient-derived fibroblasts revealed dysregulated pathways involved in osteochondrogenesis and neurodevelopment. Overall, our findings highlight a cytoplasmic role for PTBP1 in RNA stability and disease pathogenesis.

The primary cilium is a small organelle that plays key roles in cellular signaling. Defects in primary cilia formation, morphology, and function cause a heterogeneous group of developmental syndromes termed ciliopathies. The inturned planar cell polarity protein (INTU) gene acts in the CPLANE complex to facilitate ciliogenesis and support cilia signaling. Bi-allelic genetic variants in INTU have previously been reported in seven patients with pleiotropic disorders, but a core set of phenotypes from these patients has not been codified and functional studies into these variants have failed to fully demonstrate mechanistic perturbations caused by INTU dysfunction. Here, we report on a person with cardiac abnormalities, distinctive craniofacial features, developmental delays, tongue hamartomas, bilateral clinodactyly, and polydactyly of the left great toe. Trio whole-exome sequencing identified compound heterozygous variants in the INTU gene. Functional studies provide evidence that these INTU variants confer human disease through altered ciliogenesis and/or cilia signaling. Furthermore, we suggest that this study along with previous reports sufficiently establishes an association between a pleiotropic disorder and variants in the INTU gene to enhance clinical interpretation of INTU variants in future studies.

Congenital heart disease (CHD) is the most common birth defect, occurring in approximately 1% of newborns. Identification of an etiology to CHD is challenged by its genetic heterogeneity and our ongoing understanding of genes involved in its pathogenesis. A critical role for a network of transcription factors has been established in orchestrating the intricate regulation of cardiac development and its importance is emphasized by an association of these genes with CHD. We describe a de novo truncating variant in HAND2 uncovered by exome sequencing in an infant affected with CHD including pulmonary valve stenosis, thick and doming pulmonic valve, patent foramen ovale, and heart murmur. The CHD was not observed in isolation, as the proband also exhibited dysmorphic facial features, overlapping 4th and 5th toes, and slow growth. HAND2, a basic helix-loop-helix transcription factor, plays a crucial role in regulating gene expression during embryogenesis, particularly in morphogenesis of the heart and limb. Mouse models of Hand2 deficiency support this role as they exhibit congenital heart malformations and limb defects. Here, we add to the increasing body of evidence linking HAND2 to CHD and highlight a need for further investigation to elucidate the role of this transcription factor in human disease. Mechanistic insight into HAND2 variants has the potential to improve diagnostic yield and therapeutic strategies for CHD as well as other developmental anomalies.

Here we describe a neonate exhibiting hypotonia, macrocephaly, renal cysts, and respiratory failure requiring tracheostomy and ventilator support. Genetic analysis via rapid genome sequencing (rGS) identified a loss on chromosome 4 encompassing polycystin-2 (PKD2) and a loss on chromosome 22 encompassing SH3 and Multiple Ankyrin Repeat Domains 3 (SHANK3), indicative of Phelan-McDermid syndrome. Further analysis via traditional karyotyping, Optical Genome Mapping (OGM), and PacBio long-read sequencing revealed a more complex landscape of chromosomal rearrangements in this individual, including a balanced 3;12 translocation, and an unbalanced 17;22 translocation. The proband's phenotypic presentation is thought to be the result of Phelan-McDermid syndrome and represents an expansion of the described phenotypes to include significant respiratory failure. This study underscores the challenges and importance of comprehensive genetic testing in elucidating complex presentations and highlights the need for complementary testing methods to overcome limitations in resolution.

Wilms tumor (WT) is the most common renal malignancy in pediatric patients, often arising sporadically but occasionally linked to familial predisposition syndromes. Copy number losses or loss of heterozygosity (LOH) at 1p, 11p15, 16q, 17p, and/or copy number gains in 1q have been previously reported to carry prognostic significance. Since 2018, we assessed frozen or formalin-fixed paraffin-embedded (FFPE) tissue WT samples submitted for study from across the nation and around the world using a genome wide oncology-based microarray. In a subset of cases across a three-year span (n=955), 600 (63%) exhibited positive findings, 261 (27%) were negative, and 94 (10%) could not be analyzed due to insufficient tumor percentage or DNA quality/quantity.Our cumulative analyses revealed 11p15 loss/LOH to be the most recurrent finding (n=390), followed by 1q gain (n=296), 16p loss/LOH (n=157), 1p loss/LOH (n=141), and 17p loss/LOH (n=68). A single finding was observed in 304 cases, 11p15 loss/LOH (n=182), 1q gain (n=82), 16q loss/LOH (n=18), 1p loss/LOH (n=10), and 17p loss/LOH (n=12). We also observed cases with 2 abnormalities (n=168), 3 abnormalities (n=101), 4 abnormalities (n=22) and 5 abnormalities (n=5). The most observed co-occurrence was with 1q gain and 11p15 loss/LOH (n=47). We will further dissect the cases with co-occurring abnormalities and provide a summary of other frequent findings that are not currently being reported to determine if a more comprehensive genomic analysis of Wilms Tumor is warranted.

There are currently over 7,000 rare diseases estimated to affect nearly 30 million Americans, according to the National Organization for Rare Disease (NORD). A variety of genetic, biochemical, and other diagnostic tests are available to such patients. While microarrays and gene panels are often considered standard of care, clinicians are increasingly ordering exome sequencing on such patients as a first-tier diagnostic assay. Large cohort studies have shown that exome sequencing has a consistent diagnostic yield of 30-50% depending on the patient’s condition, but this still fails to provide an answer for more than half of individuals tested.

BACKGROUND:Throughout history, the field of cytogenetics has witnessed significant changes due to the constant evolution of technologies used to assess chromosome number and structure. Similar to the evolution of single nucleotide variant detection from Sanger sequencing to next-generation sequencing, the identification of chromosome alterations has progressed from banding to fluorescence in situ hybridization (FISH) to chromosomal microarrays. More recently, emerging technologies such as optical genome mapping and genome sequencing have made noteworthy contributions to clinical laboratory testing in the field of cytogenetics.CONTENT:In this review, we journey through some of the most pivotal discoveries that have shaped the development of clinical cytogenetics testing. We also explore the current test offerings, their uses and limitations, and future directions in technology advancements.SUMMARY:Cytogenetics methods, including banding and targeted assessments like FISH, continue to hold crucial roles in cytogenetic testing. These methods offer a rapid turnaround time, especially for conditions with a known etiology involving recognized cytogenetic aberrations. Additionally, laboratories have the flexibility to now employ higher-throughput methodologies to enhance resolution for cases with greater complexity.

Background: Leigh syndrome is a rare, genetic, and severe mitochondrial disorder characterized by neuromuscular issues (ataxia, seizure, hypotonia, developmental delay, dystonia) and ocular abnormalities (nystagmus, atrophy, strabismus, ptosis). It is caused by pathogenic variants in either mitochondrial or nuclear DNA genes, with an estimated incidence rate of 1 per 40,000 live births.Case presentation: Herein, we present an infant male with nystagmus, hypotonia, and developmental delay who carried a clinical diagnosis of Leigh-like syndrome. Cerebral magnetic resonance imaging changes further supported the clinical evidence of an underlying mitochondrial disorder, but extensive diagnostic testing was negative. Trio exome sequencing under a research protocol uncovered compound-heterozygous missense variants in the HTRA2 gene (MIM: #606441): NM_013247.5:c.1037A>T:(p.Glu346Val) (maternal) and NM_013247.5:c.1172T>A:(p.Val391Glu) (paternal). Both variants are absent from public databases, making them extremely rare in the population. The maternal variant is adjacent to an exon-intron boundary and predicted to disrupt splicing, while the paternal variant alters a highly conserved amino acid and is predicted to be damaging by nearly all in silico tools. Biallelic variants in HTRA2 cause 3-methylglutaconic aciduria, type VIII (MGCA8), an extremely rare autosomal recessive disorder with fewer than ten families reported to date. Variant interpretation is challenging given the paucity of known disease-causing variants, and indeed we assess both paternal and maternal variants as Variants of Uncertain Significance under current American College of Medical Genetics guidelines. However, based on the inheritance pattern, suggestive evidence of pathogenicity, and significant clinical correlation with other reported MGCA8 patients, the clinical care team considers this a diagnostic result.Conclusion: Our findings ended the diagnostic odyssey for this family and provide further insights into the genetic and clinical spectrum of this critically under-studied disorder.

Long-read sequencing can often overcome the deficiencies in routine microarray or short-read technologies in detecting complex genomic rearrangements. Here we used Pacific Biosciences circular consensus sequencing to resolve complex rearrangements in two patients with rare genetic anomalies. Copy number variants (CNVs) identified by clinical microarray -chr8p deletion and chr8q duplication in patient 1, and interstitial deletions of chr18q in patient 2-were suggestive of underlying rearrangements. Long-read genome sequencing not only confirmed these CNVs but also revealed their genomic structures. In patient 1, we resolved a novel recombinant chromosome 8 (Rec8)-like rearrangement with a 3.43 Mb chr8q terminal duplication that was linked to a 7.25-8.21 Mb chr8p terminal deletion. In patient 2, we uncovered a novel complex rearrangement involving a 1.17 Mb rearranged segment and four interstitial deletions ranging from 9 bp to 12.39 Mb. Our results underscore the diversity of clinically relevant structural rearrangements and the power of long-read sequencing in unraveling their nuanced architectures.

Abstract Background Chromosomal microarray (CMA) is commonly utilized in the obstetrics setting. CMA is recommended when one or more fetal structural abnormalities is identified. CMA is also commonly used to determine genetic etiologies for miscarriages, fetal demise, and confirming positive prenatal cell‐free DNA screening results. Methods In this study, we retrospectively examined 523 prenatal and 319 products‐of‐conception (POC) CMA cases tested at Nationwide Children's Hospital from 2011 to 2020. We reviewed the referral indications, the diagnostic yield, and the reported copy number variants (CNV) findings. Results. In our cohort, the diagnostic yield of clinically significant CNV findings for prenatal testing was 7.8% (n = 41/523) compared to POC testing (16.3%, n = 52/319). Abnormal ultrasound findings were the most common indication present in 81% of prenatal samples. Intrauterine fetal demise was the common indication identified in POC samples. The most common pathogenic finding observed in all samples was isolated trisomy 21, detected in seven samples. Conclusion Our CMA study supports the clinical utility of prenatal CMA for clinical management and identifying genetic etiology in POC arrays. In addition, it provides insight to the spectrum of prenatal and POC CMA results as detected in an academic hospital clinical laboratory setting that serves as a reference laboratory.

Introduction/AimsExome sequencing (ES) has proven to be a valuable diagnostic tool for neuromuscular disorders, which often pose a diagnostic challenge. The aims of this study were to investigate the clinical outcomes associated with utilization of ES in the pediatric neuromuscular clinic and to determine if specific phenotypic features or abnormal neurodiagnostic tests were predictive of a diagnostic result.MethodsThis was a retrospective medical record review of 76 pediatric neuromuscular clinic patients who underwent ES. Based upon clinical assessment prior to ES, patients were divided into two groups: affected by neuromuscular (n = 53) or non-neuromuscular (n = 23) syndromes.ResultsA diagnosis was made in 28/76 (36.8%), with 29 unique disorders identified. In the neuromuscular group, a neuromuscular condition was confirmed in 78% of those receiving a genetic diagnosis. Early age of symptom onset was associated with a significantly higher diagnostic yield. The most common reason neuromuscular diagnoses were not detected on prior testing was due to causative genes not being present on disease-specific panels. Changes to medical care were made in 57% of individuals receiving a diagnosis on ES.DiscussionThese data further support ES as a powerful diagnostic tool in the pediatric neuromuscular clinic and highlight the advantages of ES over gene panels, including the ability to identify diagnoses regardless of etiology, identify genes newly associated with disease, and identify multiple confounding diagnoses. Rapid and accurate diagnosis by ES can not only end the patient's diagnostic odyssey, but often impacts patients' medical management and genetic counseling of families.

Anorectal malformations (ARMs) constitute a group of congenital defects of the gastrointestinal and urogenital systems. They affect males and females, with an estimated worldwide prevalence of 1 in 5000 live births. These malformations are clinically heterogeneous and can be part of a syndromic presentation (syndromic ARM) or as a nonsyndromic entity (nonsyndromic ARM). Despite the well-recognized heritability of nonsyndromic ARM, the genetic etiology in most patients is unknown. In this study, we describe three siblings with diverse congenital anomalies of the genitourinary system, anemia, delayed milestones, and skeletal anomalies. Genome sequencing identified a novel, paternally inherited heterozygous Caudal type Homeobox 2 (CDX2) variant (c.722A > G (p.Glu241Gly)), that was present in all three affected siblings. The variant identified in this family is absent from population databases and predicted to be damaging by most in silico pathogenicity tools. So far, only two other reports implicate variants in CDX2 with ARMs. Remarkably, the individuals described in these studies had similar clinical phenotypes and genetic alterations in CDX2 CDX2 encodes a transcription factor and is considered the master regulator of gastrointestinal development. This variant maps to the homeobox domain of the encoded protein, which is critical for interaction with DNA targets. Our finding provides a potential molecular diagnosis for this family's condition and supports the role of CDX2 in anorectal anomalies. It also highlights the clinical heterogeneity and variable penetrance of ARM predisposition variants, another well-documented phenomenon. Finally, it underscores the diagnostic utility of genomic profiling of ARMs to identify the genetic etiology of these defects.

Chromosomal microarray (CMA) is a testing modality frequently used in pediatric patients; however, published data on its utilization are limited to the genetic setting. We performed a database search for all CMA testing performed from 2010 to 2020, and delineated the diagnostic yield based on patient characteristics, including sex, age, clinical specialty of providers, indication of testing, and pathogenic finding. The indications for testing were further categorized into Human Phenotype Ontology categories for analysis. This study included a cohort of 14,541 patients from 29 different medical specialties, of whom 30% were from the genetics clinic. The clinical indications for testing suggested that neonatology patients demonstrated the greatest involvement of multiorgan systems, involving the most Human Phenotype Ontology categories, compared with developmental behavioral pediatrics and neurology patients being the least. The top pathogenic findings for each specialty differed, likely due to the varying clinical features and indications for testing. Deletions involving the 22q11.21 locus were the top pathogenic findings for patients presenting to genetics, neonatology, cardiology, and surgery. Our data represent the largest pediatric cohort published to date. This study is the first to demonstrate the diagnostic utility of this assay for patients seen in the setting of different specialties, and it provides normative data of CMA results among a general pediatric population referred for testing because of variable clinical presentations.

Alterations in the TAOK1 gene have recently emerged as the cause of developmental delay with or without intellectual impairment or behavioral abnormalities (MIM # 619575). The 32 cases currently described in the literature have predominantly de novo alterations in TAOK1 and a wide spectrum of neurodevelopmental abnormalities. Here, we report four patients with novel pathogenic TAOK1 variants identified by research genome sequencing, clinical exome sequencing, and international matchmaking. The overlapping clinical features of our patients are consistent with the emerging core phenotype of TAOK1-associated syndrome: facial dysmorphism, feeding difficulties, global developmental delay, joint laxity, and hypotonia. However, behavioral abnormalities and gastrointestinal issues are more common in our cohort than previously reported. Two patients have de novo TAOK1 variants (one missense, one splice site) consistent with most known alterations in this gene. However, we also report the first sibling pair who both inherited a TAOK1 frameshift variant from a mildly affected mother. Our findings suggest that incomplete penetrance and variable expressivity are relatively common in TAOK1-associated syndrome, which holds important implications for clinical genetic testing.