Cornelia de Lange Syndrome (CdLS) is a multisystem disorder caused by pathogenic variants in one of the six genes associated with CdLS (NIPBL, SMC3, SMC1A, HDAC8, RAD21, BRD4) and pathogenic variants in phenocopy genes. We hypothesized that individuals with a clinical diagnosis of CdLS and no molecular diagnosis harbor diagnostic variants that are missed by the current standard of care, exome-focused workflows. We performed a re-analysis of the genome sequencing data from a previously published cohort of 173 individuals with a clinical diagnosis of CdLS and expanded the scope of analysis to include noncoding and structural variants. The comprehensive re-analysis revealed molecular etiologies in an additional 37 probands increasing the overall diagnostic yield from 37.5% to 57.8%. The new diagnoses were enriched for variants beyond the exonic SNVs/ indels, including cryptic non-coding variants, copy number variants, balanced rearrangements such as inversions, and variants in genes that phenocopy CdLS. Transcriptome aided re-analysis uncovered cryptic noncoding variants that lacked sufficient computational evidence for aberrant splicing and yet produced aberrantly spliced mRNA. Our results underscore the need for whole genome (and transcriptome) sequencing and a comprehensive, unbiased analytical protocol to exhaustively mine a phenotypically and genetically heterogeneous cohort to maximize its diagnostic yield.

Accurate detection of all types of mitochondrial DNA (mtDNA) variants, including single large-scale mtDNA deletions (SLSMDs) and multiple mtDNA deletions (MMDs), along with heteroplasmy quantification, is essential for Primary Mitochondrial Disease (PMD) diagnosis. This study compares amplification-free PacBio long-read sequencing (LRS) mtDNA analysis with long-range PCR-based targeted mtDNA sequencing by short-read sequencing (SRS) in terms of detection sensitivity and accuracy. In total, 17 samples, including 4 SLSMD cases (3 blood, 1 muscle), 9 MMD muscle samples, and 4 deletion-negative controls (1 blood, 3 muscle), were sequenced using the PacBio Sequel IIe. Our findings demonstrate LRS's efficacy in detecting single nucleotide variants (SNVs) and large mtDNA deletions with precise breakpoints. LRS can accurately detect and distinguish SLSMD from MMD, providing deletion heteroplasmy without the need for a second methodology. Deletion heteroplasmy computed from LRS was highly correlated with the Droplet Digital PCR (ddPCR) estimates (Pearson's r2 = 0.95). While LRS can detect SNVs with approximately 5% heteroplasmy, only variants exceeding 10% heteroplasmy can attain 100% sensitivity, specificity, and precision when compared to those previously identified through clinical testing. In conclusion, our findings establish PacBio LRS as a robust tool for comprehensive mtDNA analysis capable of accurately detecting and quantifying heteroplasmic mtDNA variants and complex deletions.

Purpose:Aicardi-Goutières syndrome (AGS) is a type I interferonopathy presently associated with nine genes. PTPN1 is a negative regulator of the interferon pathway previously associated with chronic inflammation and recently type 1 IFN autoinflammation. Methods:Genomic data from undiagnosed individuals with suspected AGS were interrogated for PTPN1 variants, and predicted loss-of-function (pLOF) and damaging missense variants in PTPN1 were sought in two additional academic databases as well as the All of Us database. Results:We identified 13 cases with ultra-rare heterozygous pLOF or highly damaging missense variants in PTPN1. Nine cases were identified in a cohort of 53 individuals (~ 17%) with clinical, imaging and persistent biochemical features of AGS. Median age of onset is 1.75 years (IQR 0.67), significantly later (p< 0.0001) than other AGS genotypes. Four additional cases were identified in academic datasets with variable clinical features suggestive of autoinflammation. Additionally, 49 individuals with ultra-rare, damaging PTPN1 variants were identified in the All of Us database, none had features suggestive of AGS, but autoimmunity was highly prevalent (~21.6%). Conclusion:Our data implicate PTPN1 as a cause of later-onset presentations of AGS within a broader spectrum of autoinflammatory phenotypes. Segregation and biobank data demonstrate reduced penetrance, with carriers being enriched for autoimmune disorders.

Pathogenic, biallelic variants in LIG3 are known to cause Mitochondrial DNA Depletion syndrome 20 with variable expression and severity. We describe a child with progressive encephalopathy, cataracts, movement disorder, endocrine dysfunction, and immunodeficiency who remained undiagnosed despite multiple negative clinical genomic diagnostic studies. Research reanalysis of PacBio long-read genome sequencing data identified compound heterozygous LIG3 variants, including a splice variant and a novel 98 bp insertion. Western blot confirmed loss of LIG3 protein expression and RNA-seq demonstrated aberrant transcripts. Muscle biopsy revealed mitochondrial dysfunction, with COX-deficient fibers and complex IV deficiency. Notably, this is the first reported association of LIG3 deficiency with immunologic and endocrine abnormalities, emphasizing the importance of a broad approach to phenotype-genotype.

Biallelic mutations in VPS45 disrupt endosomal protein trafficking, leading to a rare immunodeficiency syndrome characterized by neutrophil dysfunction, with fewer than 40 cases reported. Key features of the condition include neutropenia, recurrent infections, hepatosplenomegaly, nephromegaly, and myelofibrosis. Neurodevelopmental abnormalities, such as global developmental delay, hypotonia, nystagmus, and cortical blindness, have been associated with a specific variant c.712G>A; p.Glu238Lys. Here we present a case with presumed familial hemophagocytic lymphohistiocytosis (HLH) in infancy and progressive neurological symptoms. The patient is a 15-year-old female, who initially presented at 4 weeks of age with severe mastoiditis. She was found to have poor NK cell function, perforin deficiency, and neutropenia. Bone marrow biopsy showed toxic granulation and cytoplasmic vacuolation in some neutrophils. Few histiocytes and no hemophagocytes were seen. She underwent bone marrow transplant (BMT) at 4 months old due to presumed HLH and a history of a brother with HLH who died from an infection post-BMT. She has a history of global developmental delay, dysgraphia, ADHD, primary ovarian insufficiency, multiple fractures, atypical bony development in the feet, and hypercholesterolemia. On examination, she has short stature, facial dysmorphism, atypical dentition, nystagmus, choreiform movements, and gait ataxia. Brain imaging at 10 years of age showed basal ganglia and subcortical white matter calcifications in the frontal lobes. Extensive genetic testing, including genome sequencing and mitochondrial DNA sequencing, was negative. Research-based reanalysis revealed novel biallelic VPS45 missense variants (c.652C>T; p.Arg218Cys and c.1157G>A; p.Arg386His), confirmed by Sanger sequencing. Parental testing demonstrated the variants were inherited in trans from unaffected carriers and were also found in the deceased brother’s exome data. While a specific variant (p.Glu238Lys) has been linked to neurological symptoms in VPS45 deficiency, it was absent in our case. The patient’s progressive neurological phenotype, including chorea, basal ganglia, and subcortical white matter calcifications, suggests further phenotype expansion. The overlap with HLH and post-BMT complications underscores the diagnostic and therapeutic complexities, emphasizing the need for further research into VPS45-related pathophysiology and its clinical implications. This case expands the understanding of VPS45 deficiency by highlighting pronounced neurological involvement, which appears more striking in our proband.

A key problem in genetics is associating variants with disease phenotypes. In aid of this, much progress has been made in quantifying the functional impact of individual variants on the gene product it codes for. However, the intolerance of the sequence in which those variants are found to functional variation is also a key determinant of whether a deleterious variant is pathogenic or not. Previous approaches to estimating genic intolerance have combined functional variant types, i.e., missense, loss-of-function, etc., or restricted analyses to only one type, i.e., pLI, missense-Z etc. Here we take a different approach and jointly model patterns of intolerance across multiple functional variant types. We refer to this approach as CATMINT. We show that CATMINT is competitive with previous gene level intolerance metrics in predicting disease relevant genes, with CATMINT ranking among the top performing scores across differing types of genes. However, perhaps more exciting is that CATMINT enables variant category specific intolerance estimation, revealing distinct functional profiles across genes/gene families. Analysis of ClinVar data shows that CATMINT intolerance patterns in disease genes recapitulate patterns of pathogenic variants within those genes, supporting the utility of category-specific intolerance in clinical variant interpretation. Further, we use the statistical framework utilized by CATMINT to conduct power analyses, allowing us to classify genes according to the power those genes have to detect intolerance. This allows us, for example, to identify genes that are underpowered and undetected, but may nevertheless be highly intolerant. Together, these results define a framework for understanding how selective pressures shape gene-specific sensitivity to different classes of mutation, improving the resolution of variant interpretation and gene prioritization in clinical and functional genomics. ### Competing Interest Statement The authors have declared no competing interest.

Introduction:Molecular analysis of red cell disorders has revolutionized diagnosis, however, there remain challenges. Main Symptoms:This patient presented with hemolytic anemia in the newborn period. He required chronic transfusions to maintain his hemoglobin level until 6 years of age. A splenectomy was performed at 3 years of age. Main Diagnoses:Using whole genome sequencing, we were able to identify a duplication upstream of the red cell promoter of HK1. Long-read RNA sequencing established aberrant expression off of this promoter. Conclusions:These non-coding variants remain challenging to identify. His promoter duplication may have a founder effect in South Asia.

Accurate detection of all types of mitochondrial DNA (mtDNA) variants, including single large-scale mtDNA deletions (SLSMDs) and multiple mtDNA deletions (MMDs), along with heteroplasmy quantification, is essential for Primary Mitochondrial Disease (PMD) diagnosis. This study compares amplification-free PacBio Long Read Sequencing (LRS) mtDNA analysis with long-range PCR-based targeted mtDNA sequencing by Short Read Sequencing (SRS) in terms of detection sensitivity and accuracy. Seventeen samples, including SLSMD cases (3 blood, 2 muscle), 8 MMD muscle samples, and 4 deletion-negative controls (2 blood, 2 muscle), were sequenced using the PacBio Sequel II. Our findings demonstrate LRS’s efficacy in detecting SNVs and large mtDNA deletions with precise breakpoints. Deletion heteroplasmy computed from LRS was highly correlated with the ddPCR estimates (Pearson’s r2 = 0.95). LRS detected 100% of SNVs with heteroplasmy > 10% previously identified through clinical testing. Our findings highlight the utility of PacBio LRS as a valuable tool for advanced mtDNA analysis.

Mitochondrial DNA depletion syndrome 20 (MTDPS20) is a rare disorder caused by variants in the LIG3 gene, which encodes a DNA ligase essential for mitochondrial DNA repair and replication. Defects in LIG3 impair mitochondrial replication, leading to reduced mitochondrial DNA copy numbers and mitochondrial dysfunction. Since its description in 2021, fewer than 20 cases have been reported, with features including gastrointestinal dysmotility, leukoencephalopathy, muscle weakness, neurogenic bladder, and cognitive decline. A more severe phenotype, including progressive myopathy, fatal muscle degeneration, decreased cytochrome c oxidase (COX) activity, and lipid accumulation in muscle fibers, was also reported. Here, we report a case of MTDPS20 with biallelic LIG3 variants that presented with immunodeficiency as part of the clinical phenotype for the first time. The patient was a 23-month-old female with a history of developmental delay, infantile spasms, sensorineural hearing loss, bilateral cataracts, optic nerve hypoplasia, failure to thrive, gastroesophageal reflux, gastrointestinal dysmotility, anhidrosis, and progressive encephalopathy. On examination, she had severe hypotonia with no head control, microcephaly, and choreiform movements. Immune findings included hypogammaglobulinemia requiring subcutaneous immunoglobulin therapy, while endocrine features included central hypothyroidism and type 1 diabetes mellitus. Muscle biopsy demonstrated COX-negative muscle fibers with lipid accumulation. Neuroimaging demonstrated mildly delayed myelination and volume loss. The patient died at age 4 due to respiratory distress. Comprehensive genetic testing, including exome sequencing, RNA sequencing, genome sequencing, and metabolomics, was uninformative. Reanalysis of genome sequencing data identified biallelic LIG3 variants: a missense mutation (c.1209-2A>G) and a 93-bp insertion, each inherited from unaffected parents. Functional validation via western blot and Sanger sequencing confirmed the pathogenic nature of these variants. This case includes the first report of immune dysfunction in MTDPS20, characterized by hypogammaglobulinemia and type 1 diabetes mellitus, suggesting involvement of immune dysregulation. These findings, alongside typical manifestations such as gastrointestinal dysmotility, leukoencephalopathy, and COX-negative fibers, expand the recognized phenotype of MTDPS20 to include immunodeficiency. Immunodeficiency is identified as a feature of MTDPS20 in this case, expanding the clinical spectrum of the disorder. Further research is needed to investigate the underlying mechanisms and implications for diagnosis and management.

Purpose: We developed a genome sequencing-based test (Rapid Targeted Analysis of the Genome for Infants [rTAG-I]) to minimize turnaround time while maximizing diagnostic yield and access to rapid sequencing for critically ill infants. We sought to create a system of predicting which infants would have a molecular finding. Methods: We performed a prospective observational study of infants referred for genetics consult who received rTAG-I testing, which analyzes 3183 curated genes with phenotype-agnostic prioritization of pathogenic and likely pathogenic variants. Infants were stratified by perceived likelihood of a diagnostic result and divided into “Likely,” “Uncertain,” and “Not Likely.” We also assessed whether reportable findings correlated with patient phenotypes. Results: We identified reportable findings in 133/400 (33%) infants. Access to rapid testing increased from 1% to 20% of all infants hospitalized in the neonatal/infant intensive care unit and cardiac intensive care unit, with a median turnaround time of 4.9 days. rTAG-I performed as well as exome/genome sequencing. Clinically associated results were identified in 59% of the “Likely” group and 9% of the “Not Likely” group. Conclusion: rTAG-I produced a high rate of reportable findings with a rapid turnaround time. Our ability to predict infants who would benefit most was imperfect, reinforcing that broad access to genome-based testing is still required.

Background and Objectives:Canavan disease (CD) is a neurodegenerative disorder in which biallelic pathogenic variants in ASPA result in spongiform degeneration of the cerebral white matter, leading to progressive and irreversible motor and cognitive decline. Despite comprehensive genetic testing, many individuals with clinical and biochemical diagnoses of CD remain without a definitive molecular diagnosis. This gap hinders access to emerging gene-targeted therapies and limits participation in clinical trials. Our objective was to understand the genetic etiology of 8 unsolved cases of CD. Methods:We used long-read sequencing (LRS) to investigate 8 individuals clinically and biochemically diagnosed with CD but who had negative genetic testing results. We performed targeted LRS using the Oxford Nanopore Technologies platform for 3 unrelated individuals and PacBio HiFi for an additional individual from our cohort. We performed targeted LRS on barcoded and pooled samples from the remaining affected individuals. To investigate functional impact on gene function, we performed RNA sequencing (RNA-seq) with and without cycloheximide on fibroblasts. We then evaluated the allele frequency in the population using gnomAD. Results:We identified an ∼2,600-bp SVA_E retrotransposon intronic insertion in ASPA in all 8 individuals. The insertion was found to be either homozygous or compound heterozygous trans with a known pathogenic variant in all individuals. RNA-seq indicated that the SVA_E insertion creates a novel splice acceptor site within intron 4 of ASPA that causes aberrant splicing and transcript degradation. Surprisingly, the frequency of this variant in population databases suggests that it is the most common pathogenic variant in ASPA and that it is present across ancestry groups. Discussion:Our study identified the most common pathogenic variant in ASPA, which has been overlooked in 25 years of CD research. Considering this, it is important to ensure that all testing laboratories can detect this variant through diagnostic testing and carrier screening. Our study highlights a substantial blind spot in standard short-read diagnostic pipelines, which historically have missed or overlooked these types of insertions. It also shows the power of emerging technologies, such as LRS and RNA-seq, to identify new classes of variants for genetic disorders, including CD.

The use of genetics has transformed nearly every medical specialty. Allergy and immunology are no exceptions, and finding a way to harness data of this type requires mastery of some new skills to serve our patients. This review covers who should be tested, what testing is appropriate, how to interpret of results, and how to apply the results for patient management. Additional case studies with step-by-step workflows and other resources are provided in the Supplemental Methods (available in the Online Repository at www.jacionline.org).

Background:Genetic testing is essential to the diagnosis of nonsyndromic bilateral sensorineural hearing loss (BSNHL), where pathogenic variants in GJB2 are the most common cause. Current testing strategies often fail to provide a comprehensive diagnosis and typically require the use of multiple testing methodologies. This study evaluated the diagnostic utility of genome sequencing (GS) in a cohort with heterozygosity for GJB2 pathogenic variants and BSNHL. Methods:A retrospective cohort of 23 individuals with BSNHL and a heterozygous pathogenic variant in GJB2 underwent targeted GJB2 resequencing and variant reinterpretation. Those without biallelic GJB2 variants upon single gene reanalysis proceeded to exome sequencing (ES) using a large virtual panel of hearing loss-associated genes. Subjects with no definitive diagnosis from ES subsequently underwent GS. Variants were interpreted using hearing loss-specific ACMG guidelines and published literature. Results:Three individuals were diagnosed with biallelic pathogenic variants upon GJB2 single gene reanalysis. ES identified a definitive or likely diagnosis in five different hearing loss-related genes in 5/20 (25%) individuals, while two additional cases remained inconclusive due to novel or ambiguous variants in two other hearing loss-associated genes. GS of the remaining 15 cases yielded diagnoses in three individuals, including the identification of deletions in LOXHD1 and STRC, and a recently characterized 125 kb deletion overlapping CRYL1, which refines a critical upstream regulatory region associated with GJB2-related hearing loss. Overall, 11/23 (48%) individuals received a diagnosis with our stepwise testing approach, with GS providing sequencing coverage of all findings. Conclusion:GS improves diagnostic yield in patients with BSNHL, capturing both SNVs and CNVs missed by ES and targeted testing, and supports its adoption as a comprehensive first-tier diagnostic test for nonsyndromic hearing loss.

Background Placental malperfusion (PMP), a vascular disorder of impaired maternal or fetal placental perfusion, is common in fetuses with congenital heart disease (CHD), yet its impact on outcomes and underlying mechanisms are unclear. Perturbation of shared developmental pathways between the fetal heart and placenta may play a role in PMP development. Objective This study sought to determine whether CHD fetuses with PMP have adverse outcomes and genomic variation in vascular or placental developmental pathways. Methods This was a retrospective case-control study of 299 CHD fetuses with and without PMP. Maternal, genomic, placental, and outcomes data were reviewed. Placentas with PMP met pathologic criteria for maternal or fetal vascular malperfusion (Amsterdam criteria), or placental weight <10%. Rare, predicted deleterious exonic/putative splicing single nucleotide variants, small insertion/deletions, and de novo variants from trios were identified from genome sequencing data, followed by burden analysis and pathway enrichment. Mann-Whitney and Student’s-t test or chi-square and Fisher exact tests were performed. Results PMP was present in 51% of fetuses. Nonsyndromic fetuses with PMP (n = 122) vs without PMP (n = 124) had decreased birth weight (2,986 g vs 3,330 g, P < 0.001), length (48 cm vs 48.9 cm, P < 0.001), and head circumference (33 cm vs 34 cm, P < 0.001); a longer index hospitalization (21 days vs 15 days, P = 0.04); and a trend toward increased mortality (12.3% vs 5.6%, P = 0.07). No significant differences were found in syndromic subjects. PMP was associated with increased mortality in univariable analysis (unadjusted HR: 1.5; 95% CI: 1.03-2.17; P = 0.03), with borderline significance in multivariable analysis (adjusted HR: 1.44; 95% CI: 0.995-2.09; P = 0.054). The proportion of fetuses with and without PMP with ≥1 angiogenesis pathway or de novo variant was similar and not associated with outcomes. All fetuses with PMP demonstrated distinct enrichment in de novo variants in Notch signaling (NOTCH1, NOTCH3, DLL4, SMAD6, MMP2, ID2, SOX9, CHD7) and extracellular matrix regulation and epithelial-to-mesenchymal transition (COL4A4, FN1, MMP2, VIM, TIE1, DSP, PUF60, HNRNPK, SPON1) in genes critical for trophoblast and placental vascular development. Conclusion PMP is associated with impaired fetal growth, longer hospitalization, and possibly increased mortality in CHD. CHD fetuses with PMP harbor deleterious de novo variants in several key placental and cardiac developmental pathways, which may represent important mechanisms underlying PMP in CHD.