Expanding the genetics and phenotypes of ocular congenital cranial dysinnervation disorders.

Purpose: To identify genetic etiologies and genotype/phenotype associations for unsolved ocular congenital cranial dysinnervation disorders (oCCDDs). Methods: We coupled phenotyping with exome or genome sequencing of 467 pedigrees with genetically unsolved oCCDDs, integrating analyses of pedigrees, human and animal model phenotypes, and de novo variants to identify rare candidate single nucleotide variants, insertion/deletions, and structural variants disrupting protein-coding regions. Prioritized variants were classified for pathogenicity and evaluated for genotype/phenotype correlations. Results: Analyses elucidated phenotypic subgroups, identified pathogenic/likely pathogenic variant(s) in 43/467 probands (9.2%), and prioritized variants of uncertain significance in 70/467 additional probands (15.0%). These included known and novel variants in established oCCDD genes, genes associated with syndromes that sometimes include oCCDDs (e.g., MYH10, KIF21B, TGFBR2, TUBB6), genes that fit the syndromic component of the phenotype but had no prior oCCDD association (e.g., CDK13, TGFB2), genes with no reported association with oCCDDs or the syndromic phenotypes (e.g., TUBA4A, KIF5C, CTNNA1, KLB, FGF21), and genes associated with oCCDD phenocopies that had resulted in misdiagnoses. Conclusion: This study suggests that unsolved oCCDDs are clinically and genetically heterogeneous disorders often overlapping other Mendelian conditions and nominates many candidates for future replication and functional studies.

Mono and biallelic variants in HCN2 cause severe neurodevelopmental disorders.

Hyperpolarization activated Cyclic Nucleotide (HCN) gated channels are crucial for various neurophysiological functions, including learning and sensory functions, and their dysfunction are responsible for brain disorders, such as epilepsy. To date, HCN2 variants have only been associated with mild epilepsy and recently, one monoallelic missense variant has been linked to developmental and epileptic encephalopathy. Here, we expand the phenotypic spectrum of HCN2- related disorders by describing twenty-one additional individuals from fifteen unrelated families carrying HCN2 variants. Seventeen individuals had developmental delay/intellectual disability (DD/ID), two had borderline DD/ID, and one had borderline DD. Ten individuals had epilepsy with DD/ID, with median age of onset of 10 months, and one had epilepsy with normal development. Molecular diagnosis identified thirteen different pathogenic HCN2 variants, including eleven missense variants affecting highly conserved amino acids, one frameshift variant, and one in-frame deletion. Seven variants were monoallelic of which five occurred de novo, one was not maternally inherited, one was inherited from a father with mild learning disabilities, and one was of unknown inheritance. The remaining six variants were biallelic, with four homozygous and two compound heterozygous variants. Functional studies using two-electrode voltage-clamp recordings in Xenopus laevis oocytes were performed on three monoallelic variants, p.(Arg324His), p.(Ala363Val), and p.(Met374Leu), and three biallelic variants, p.(Leu377His), p.(Pro493Leu) and p.(Gly587Asp). The p.(Arg324His) variant induced a strong increase of HCN2 conductance, while p.(Ala363Val) and p.(Met374Leu) displayed dominant negative effects, leading to a partial loss of HCN2 channel function. By confocal imaging, we found that the p.(Leu377His), p.(Pro493Leu) and p.(Gly587Asp) pathogenic variants impaired membrane trafficking, resulting in a complete loss of HCN2 elicited currents in Xenopus oocytes. Structural 3D-analysis in depolarized and hyperpolarized states of HCN2 channels, revealed that the pathogenic variants p.(His205Gln), p.(Ser409Leu), p.(Arg324Cys), p.(Asn369Ser) and p.(Gly460Asp) modify molecular interactions altering HCN2 function. Taken together, our data broadens the clinical spectrum associated with HCN2 variants, and disclose that HCN2 is involved in developmental encephalopathy with or without epilepsy.

Functional associations of evolutionarily recent human genes exhibit sensitivity to the 3D genome landscape and disease.

Genome organization is intricately tied to regulating genes and associated cell fate decisions. In this study, we examine the positioning and functional significance of human genes, grouped by their evolutionary age, within the 3D organization of the genome. We reveal that genes of different evolutionary origin have distinct positioning relationships with both domains and loop anchors, and remarkably consistent relationships with boundaries across cell types. While the functional associations of each group of genes are primarily cell type-specific, such associations of conserved genes maintain greater stability across 3D genomic features and disease than recently evolved genes. Furthermore, the expression of these genes across various tissues follows an evolutionary progression, such that RNA levels increase from young genes to ancient genes. Thus, the distinct relationships of gene evolutionary age, function, and positioning within 3D genomic features contribute to tissue-specific gene regulation in development and disease.

The landscape of regional missense mutational intolerance quantified from 125,748 exomes.

Missense variants can have a range of functional impacts depending on factors such as the specific amino acid substitution and location within the gene. To interpret their deleteriousness, studies have sought to identify regions within genes that are specifically intolerant of missense variation 1-12 . Here, we leverage the patterns of rare missense variation in 125,748 individuals in the Genome Aggregation Database (gnomAD) 13 against a null mutational model to identify transcripts that display regional differences in missense constraint. Missense-depleted regions are enriched for ClinVar 14 pathogenic variants, de novo missense variants from individuals with neurodevelopmental disorders (NDDs) 15,16 , and complex trait heritability. Following ClinGen calibration recommendations for the ACMG/AMP guidelines, we establish that regions with less than 20% of their expected missense variation achieve moderate support for pathogenicity. We create a missense deleteriousness metric (MPC) that incorporates regional constraint and outperforms other deleteriousness scores at stratifying case and control de novo missense variation, with a strong enrichment in NDDs. These results provide additional tools to aid in missense variant interpretation.

Genome and RNA sequencing boost neuromuscular diagnoses to 62% from 34% with exome sequencing alone.

OBJECTIVE: Most families with heritable neuromuscular disorders do not receive a molecular diagnosis. Here we evaluate diagnostic utility of exome, genome, RNA sequencing, and protein studies and provide evidence-based recommendations for their integration into practice. METHODS: In total, 247 families with suspected monogenic neuromuscular disorders who remained without a genetic diagnosis after standard diagnostic investigations underwent research-led massively parallel sequencing: neuromuscular disorder gene panel, exome, genome, and/or RNA sequencing to identify causal variants. Protein and RNA studies were also deployed when required. RESULTS: Integration of exome sequencing and auxiliary genome, RNA and/or protein studies identified causal or likely causal variants in 62% (152 out of 247) of families. Exome sequencing alone informed 55% (83 out of 152) of diagnoses, with remaining diagnoses (45%; 69 out of 152) requiring genome sequencing, RNA and/or protein studies to identify variants and/or support pathogenicity. Arrestingly, novel disease genes accounted for <4% (6 out of 152) of diagnoses while 36.2% of solved families (55 out of 152) harbored at least one splice-altering or structural variant in a known neuromuscular disorder gene. We posit that contemporary neuromuscular disorder gene-panel sequencing could likely provide 66% (100 out of 152) of our diagnoses today. INTERPRETATION: Our results emphasize thorough clinical phenotyping to enable deep scrutiny of all rare genetic variation in phenotypically consistent genes. Post-exome auxiliary investigations extended our diagnostic yield by 81% overall (34-62%). We present a diagnostic algorithm that details deployment of genomic and auxiliary investigations to obtain these diagnoses today most effectively. We hope this provides a practical guide for clinicians as they gain greater access to clinical genome and transcriptome sequencing.

Neurodevelopmental disorders associated variants in ADAT3 disrupt the activity of the ADAT2/ADAT3 tRNA deaminase complex and impair neuronal migration.

The ADAT2/ADAT3 complex catalyzes the adenosine to inosine modification at the wobble position of eukaryotic tRNAs. Mutations in ADAT3 , the catalytically inactive subunit of the ADAT2/ADAT3 complex, have been identified in patients presenting with severe neurodevelopmental disorders (NDDs). Yet, the physiological function of ADAT2/ADAT3 complex during brain development remains totally unknown. Here we showed that maintaining a proper level of ADAT2/ADAT3 catalytic activity is required for correct radial migration of projection neurons in the developing mouse cortex. In addition, we not only reported 7 new NDD patients carrying biallelic variants in ADAT3 but also deeply characterize the impact of those variants on ADAT2/ADAT3 structure, biochemical properties, enzymatic activity and tRNAs editing and abundance. We demonstrated that all the identified variants alter both the expression and the activity of the complex leading to a significant decrease of I 34 with direct consequence on their steady-state. Using in vivo complementation assays, we correlated the severity of the migration phenotype with the degree of the loss of function caused by the variants. Altogether, our results indicate a critical role of ADAT2/ADAT3 during cortical development and provide cellular and molecular insights into the pathogenicity of ADAT3-related neurodevelopmental disorder.

Assessment of the evidence yield for the calibrated PP3/BP4 computational recommendations.

Purpose: To investigate the number of rare missense variants observed in human genome sequences by ACMG/AMP PP3/BP4 evidence strength, following the calibrated PP3/BP4 computational recommendations. Methods: Missense variants from the genome sequences of 300 probands from the Rare Genomes Project with suspected rare disease were analyzed using computational prediction tools able to reach PP3_Strong and BP4_Moderate evidence strengths (BayesDel, MutPred2, REVEL, and VEST4). The numbers of variants at each evidence strength were analyzed across disease-associated genes and genome-wide. Results: From a median of 75.5 rare (≤1% allele frequency) missense variants in disease-associated genes per proband, a median of one reached PP3_Strong, 3-5 PP3_Moderate, and 3-5 PP3_Supporting. Most were allocated BP4 evidence (median 41-49 per proband) or were indeterminate (median 17.5-19 per proband). Extending the analysis to all protein-coding genes genome-wide, the number of PP3_Strong variants increased approximately 2.6-fold compared to disease-associated genes, with a median per proband of 1-3 PP3_Strong, 8-16 PP3_Moderate, and 10-17 PP3_Supporting. Conclusion: A small number of variants per proband reached PP3_Strong and PP3_Moderate in 3,424 disease-associated genes, and though not the intended use of the recommendations, also genome-wide. Use of PP3/BP4 evidence as recommended from calibrated computational prediction tools in the clinical diagnostic laboratory is unlikely to inappropriately contribute to the classification of an excessive number of variants as Pathogenic or Likely Pathogenic by ACMG/AMP rules.

Novel syndromic neurodevelopmental disorder caused by de novo deletion of CHASERR, a long noncoding RNA.

Genes encoding long non-coding RNAs (lncRNAs) comprise a large fraction of the human genome, yet haploinsufficiency of a lncRNA has not been shown to cause a Mendelian disease. CHASERR is a highly conserved human lncRNA adjacent to CHD2-a coding gene in which de novo loss-of-function variants cause developmental and epileptic encephalopathy. Here we report three unrelated individuals each harboring an ultra-rare heterozygous de novo deletion in the CHASERR locus. We report similarities in severe developmental delay, facial dysmorphisms, and cerebral dysmyelination in these individuals, distinguishing them from the phenotypic spectrum of CHD2 haploinsufficiency. We demonstrate reduced CHASERR mRNA expression and corresponding increased CHD2 mRNA and protein in whole blood and patient-derived cell lines-specifically increased expression of the CHD2 allele in cis with the CHASERR deletion, as predicted from a prior mouse model of Chaserr haploinsufficiency. We show for the first time that de novo structural variants facilitated by Alu-mediated non-allelic homologous recombination led to deletion of a non-coding element (the lncRNA CHASERR) to cause a rare syndromic neurodevelopmental disorder. We also demonstrate that CHD2 has bidirectional dosage sensitivity in human disease. This work highlights the need to carefully evaluate other lncRNAs, particularly those upstream of genes associated with Mendelian disorders.

Detecting missed diagnoses of spinal muscular atrophy in genome, exome, and panel sequencing datasets.

Spinal muscular atrophy (SMA) is a genetic disorder that causes progressive degeneration of lower motor neurons and the subsequent loss of muscle function throughout the body. It is the second most common recessive disorder in individuals of European descent and is present in all populations. Accurate tools exist for diagnosing SMA from short read and long read genome sequencing data. However, there are no publicly available tools for GRCh38-aligned data from panel or exome sequencing assays which continue to be used as first line tests for neuromuscular disorders. We therefore developed and extensively validated a new tool - SMA Finder - that can diagnose SMA not only in genome, but also exome and targeted sequencing samples aligned to GRCh37, GRCh38, or T2T-CHM13. It works by evaluating aligned reads that overlap the c.840 position of SMN1 and SMN2 in order to detect the most common molecular causes of SMA. We applied SMA Finder to 16,626 exomes and 3,911 genomes from heterogeneous rare disease cohorts sequenced at the Broad Institute Center for Mendelian Genomics as well as 1,157 exomes and 8,762 targeted sequencing samples from Tartu University Hospital. SMA Finder correctly identified all 16 known SMA cases and reported nine novel diagnoses which have since been confirmed by clinical testing, with another four novel diagnoses undergoing validation. Notably, out of the 29 total SMA positive cases, 21 had an initial clinical diagnosis of muscular dystrophy, congenital myasthenic syndrome, or congenital myopathy. This underscored the frequency with which SMA can be misdiagnosed as other neuromuscular disorders and confirmed the utility of using SMA Finder to reanalyze phenotypically diverse neuromuscular disease cohorts. Finally, we evaluated SMA Finder on 198,868 individuals that had both exome and genome sequencing data within the UK Biobank (UKBB) and found that SMA Finder's overall false positive rate was less than 1 / 200,000 exome samples, and its positive predictive value (PPV) was 96%. We also observed 100% concordance between UKBB exome and genome calls. This analysis showed that, even though it is located within a segmental duplication, the most common causal variant for SMA can be detected with comparable accuracy to monogenic disease variants in non-repetitive regions. Additionally, the high PPV demonstrated by SMA Finder, the existence of treatment options for SMA in which early diagnosis is imperative for therapeutic benefit, as well as widespread availability of clinical confirmatory testing for SMA, may warrant the addition of SMN1 to the ACMG list of genes with reportable secondary findings after genome and exome sequencing.

Recurring homozygous ACTN2 variant (p.Arg506Gly) causes a recessive myopathy.

OBJECTIVE: ACTN2, encoding alpha-actinin-2, is essential for cardiac and skeletal muscle sarcomeric function. ACTN2 variants are a known cause of cardiomyopathy without skeletal muscle involvement. Recently, specific dominant monoallelic variants were reported as a rare cause of core myopathy of variable clinical onset, although the pathomechanism remains to be elucidated. The possibility of a recessively inherited ACTN2-myopathy has also been proposed in a single series. METHODS: We provide clinical, imaging, and histological characterization of a series of patients with a novel biallelic ACTN2 variant. RESULTS: We report seven patients from five families with a recurring biallelic variant in ACTN2: c.1516A>G (p.Arg506Gly), all manifesting with a consistent phenotype of asymmetric, progressive, proximal, and distal lower extremity predominant muscle weakness. None of the patients have cardiomyopathy or respiratory insufficiency. Notably, all patients report Palestinian ethnicity, suggesting a possible founder ACTN2 variant, which was confirmed through haplotype analysis in two families. Muscle biopsies reveal an underlying myopathic process with disruption of the intermyofibrillar architecture, Type I fiber predominance and atrophy. MRI of the lower extremities demonstrate a distinct pattern of asymmetric muscle involvement with selective involvement of the hamstrings and adductors in the thigh, and anterior tibial group and soleus in the lower leg. Using an in vitro splicing assay, we show that c.1516A>G ACTN2 does not impair normal splicing. INTERPRETATION: This series further establishes ACTN2 as a muscle disease gene, now also including variants with a recessive inheritance mode, and expands the clinical spectrum of actinopathies to adult-onset progressive muscle disease.

A gene pathogenicity tool "GenePy" identifies missed biallelic diagnoses in the 100,000 Genomes Project.

PURPOSE: The 100,000 Genomes Project diagnosed a quarter of affected participants, but 26% of diagnoses were not on the applied gene panel(s); with many being de novo variants. Assessing biallelic variants without a gene panel is more challenging. METHODS: We sought to identify missed biallelic diagnoses using GenePy, which incorporates allele frequency, zygosity, and a user-defined deleterious metric, generating an aggregate GenePy score per gene, per participant. We calculated GenePy scores for 2862 recessive disease genes in 78,216 100,000 Genomes Project participants. For each gene, we ranked participant GenePy scores and scrutinized affected participants without a diagnosis, whose scores ranked among the top 5 for each gene. In cases which participant phenotypes overlapped with the disease gene of interest, we extracted rare variants and applied phase, ClinVar, and ACMG classification. RESULTS: 3184 affected individuals without a molecular diagnosis had a top-5-ranked GenePy score and 682 of 3184 (21%) had phenotypes overlapping with a top-ranking gene. In 122 of 669 (18%) phenotype-matched cases (excluding 13 withdrawn participants), we identified a putative missed diagnosis (2.2% of all undiagnosed participants). A further 334 of 669 (50%) cases have a possible missed diagnosis but require functional validation. CONCLUSION: Applying GenePy at scale has identified 456 potential diagnoses, demonstrating the value of novel diagnostic strategies.

Inferring compound heterozygosity from large-scale exome sequencing data.

Recessive diseases arise when both copies of a gene are impacted by a damaging genetic variant. When a patient carries two potentially causal variants in a gene, accurate diagnosis requires determining that these variants occur on different copies of the chromosome (that is, are in trans) rather than on the same copy (that is, in cis). However, current approaches for determining phase, beyond parental testing, are limited in clinical settings. Here we developed a strategy for inferring phase for rare variant pairs within genes, leveraging genotypes observed in the Genome Aggregation Database (v2, n = 125,748 exomes). Our approach estimates phase with 96% accuracy, both in trio data and in patients with Mendelian conditions and presumed causal compound heterozygous variants. We provide a public resource of phasing estimates for coding variants and counts per gene of rare variants in trans that can aid interpretation of rare co-occurring variants in the context of recessive disease.

A genomic mutational constraint map using variation in 76,156 human genomes.

The depletion of disruptive variation caused by purifying natural selection (constraint) has been widely used to investigate protein-coding genes underlying human disorders1-4, but attempts to assess constraint for non-protein-coding regions have proved more difficult. Here we aggregate, process and release a dataset of 76,156 human genomes from the Genome Aggregation Database (gnomAD)-the largest public open-access human genome allele frequency reference dataset-and use it to build a genomic constraint map for the whole genome (genomic non-coding constraint of haploinsufficient variation (Gnocchi)). We present a refined mutational model that incorporates local sequence context and regional genomic features to detect depletions of variation. As expected, the average constraint for protein-coding sequences is stronger than that for non-coding regions. Within the non-coding genome, constrained regions are enriched for known regulatory elements and variants that are implicated in complex human diseases and traits, facilitating the triangulation of biological annotation, disease association and natural selection to non-coding DNA analysis. More constrained regulatory elements tend to regulate more constrained protein-coding genes, which in turn suggests that non-coding constraint can aid the identification of constrained genes that are as yet unrecognized by current gene constraint metrics. We demonstrate that this genome-wide constraint map improves the identification and interpretation of functional human genetic variation.

A Panel-Agnostic Strategy 'HiPPo' Improves Diagnostic Efficiency in the UK Genomic Medicine Service.

Genome sequencing is available as a clinical test in the UK through the Genomic Medicine Service (GMS). The GMS analytical strategy predominantly filters genome data on preselected gene panels. Whilst this reduces variants requiring assessment by reporting laboratories, pathogenic variants outside applied panels may be missed, and variants in genes without established disease-gene relationships are largely ignored. This study compares the analysis of a research exome to a GMS clinical genome for the same patients. For the research exome, we applied a panel-agnostic approach filtering for variants with High Pathogenic Potential (HiPPo) using ClinVar, allele frequency, and in silico prediction tools. We then restricted HiPPo variants to Gene Curation Coalition (GenCC) disease genes. These results were compared with the GMS genome panel-based approach. Twenty-four participants from eight families underwent parallel research exome and GMS genome sequencing. Exome HiPPo analysis identified a similar number of variants as the GMS panel-based approach. GMS genome analysis returned two pathogenic variants and one de novo variant. Exome HiPPo analysis returned the same variants plus an additional pathogenic variant and three further de novo variants in novel genes, where case series are underway. When HiPPo was restricted to GenCC disease genes, statistically fewer variants required assessment to identify more pathogenic variants than reported by the GMS, giving a diagnostic rate per variant assessed of 20% for HiPPo versus 3% for the GMS. With UK plans to sequence 5 million genomes, strategies are needed to optimise genome analysis beyond gene panels whilst minimising the burden of variants requiring clinical assessment.

Heterozygous loss-of-function SMC3 variants are associated with variable and incompletely penetrant growth and developmental features.

Heterozygous missense variants and in-frame indels in SMC3 are a cause of Cornelia de Lange syndrome (CdLS), marked by intellectual disability, growth deficiency, and dysmorphism, via an apparent dominant-negative mechanism. However, the spectrum of manifestations associated with SMC3 loss-of-function variants has not been reported, leading to hypotheses of alternative phenotypes or even developmental lethality. We used matchmaking servers, patient registries, and other resources to identify individuals with heterozygous, predicted loss-of-function (pLoF) variants in SMC3, and analyzed population databases to characterize mutational intolerance in this gene. Here, we show that SMC3 behaves as an archetypal haploinsufficient gene: it is highly constrained against pLoF variants, strongly depleted for missense variants, and pLoF variants are associated with a range of developmental phenotypes. Among 13 individuals with SMC3 pLoF variants, phenotypes were variable but coalesced on low growth parameters, developmental delay/intellectual disability, and dysmorphism reminiscent of atypical CdLS. Comparisons to individuals with SMC3 missense/in-frame indel variants demonstrated a milder presentation in pLoF carriers. Furthermore, several individuals harboring pLoF variants in SMC3 were nonpenetrant for growth, developmental, and/or dysmorphic features, some instead having intriguing symptomatologies with rational biological links to SMC3 including bone marrow failure, acute myeloid leukemia, and Coats retinal vasculopathy. Analyses of transcriptomic and epigenetic data suggest that SMC3 pLoF variants reduce SMC3 expression but do not result in a blood DNA methylation signature clustering with that of CdLS, and that the global transcriptional signature of SMC3 loss is model-dependent. Our finding of substantial population-scale LoF intolerance in concert with variable penetrance in subjects with SMC3 pLoF variants expands the scope of cohesinopathies, informs on their allelic architecture, and suggests the existence of additional clearly LoF-constrained genes whose disease links will be confirmed only by multi-layered genomic data paired with careful phenotyping.

Identification of a de novo mutation in TLK1 associated with a neurodevelopmental disorder and immunodeficiency.

Background: The Tousled-like kinases 1 and 2 (TLK1/TLK2) regulate DNA replication, repair and chromatin maintenance. TLK2 variants are associated with 'Intellectual Disability, Autosomal Dominant 57' (MRD57), a neurodevelopmental disorder (NDD) characterized by intellectual disability (ID), autism spectrum disorder (ASD) and microcephaly. Several TLK1 variants have been reported in NDDs but their functional significance is unknown. Methods: A male patient presenting with ID, seizures, global developmental delay, hypothyroidism, and primary immunodeficiency was determined to have a novel, heterozygous variant in TLK1 (c.1435C>G, p.Q479E) by genome sequencing (GS). Single cell gel electrophoresis, western blot, flow cytometry and RNA-seq were performed in patient-derived lymphoblast cell lines. In silico, biochemical and proteomic analysis were used to determine the functional impact of the p.Q479E variant and previously reported NDD-associated TLK1 variant, p.M566T. Results: Transcriptome sequencing in patient-derived cells confirmed expression of TLK1 transcripts carrying the p.Q479E variant and revealed alterations in genes involved in class switch recombination and cytokine signaling. Cells expressing the p.Q479E variant exhibited reduced cytokine responses and higher levels of spontaneous DNA damage but not increased sensitivity to radiation or DNA repair defects. The p.Q479E and p.M566T variants impaired kinase activity but did not strongly alter localization or proximal protein interactions. Conclusion: Our study provides the first functional characterization of TLK1 variants associated with NDDs and suggests potential involvement in central nervous system and immune system development. Our results indicate that, like TLK2 variants, TLK1 variants may impact development in multiple tissues and should be considered in the diagnosis of rare NDDs.

Classical phenylketonuria presenting as maternal PKU syndrome in the offspring of an intellectually normal woman.

Phenylketonuria (PKU) is an autosomal recessive inborn error of metabolism resulting from a deficiency of phenylalanine hydroxylase (PAH). If untreated by dietary restriction of phenylalanine intake, impaired postnatal cognitive development results from the neurotoxic effects of excessive phenylalanine (Phe). Signs and symptoms include severe intellectual disability and behavior problems with a high frequency of seizures and variable microcephaly. Maternal PKU syndrome refers to fetal damage resulting in congenital abnormalities when the mother has untreated PKU during pregnancy. Here, we report an intellectually normal 32-year-old female who presented with recurrent pregnancy loss and two neonatal deaths with congenital heart disease, microcephaly, intrauterine growth restriction, and respiratory distress. She was diagnosed with PKU through exome sequencing performed for carrier testing with a homozygous pathogenic variant in the PAH gene, c.169_171del, p.(Glu57del) that is associated with classical PKU. Consistent with the genetic finding, she had a markedly increased plasma phenylalanine concentration of 1642 µmol/L (normal <100). This case demonstrates that recurrent pregnancy loss due to untreated maternal PKU may present as an initial finding in otherwise unsuspected classical PKU and illustrates that extreme degrees of variable expressivity may occur in classical PKU. Moreover, this case illustrates the value of genomic sequencing of women who experience recurrent pregnancy loss or neonatal anomalies.

Advanced variant classification framework reduces the false positive rate of predicted loss-of-function variants in population sequencing data.

Predicted loss of function (pLoF) variants are often highly deleterious and play an important role in disease biology, but many pLoF variants may not result in loss of function (LoF). Here we present a framework that advances interpretation of pLoF variants in research and clinical settings by considering three categories of LoF evasion: (1) predicted rescue by secondary sequence properties, (2) uncertain biological relevance, and (3) potential technical artifacts. We also provide recommendations on adjustments to ACMG/AMP guidelines' PVS1 criterion. Applying this framework to all high-confidence pLoF variants in 22 genes associated with autosomal-recessive disease from the Genome Aggregation Database (gnomAD v.2.1.1) revealed predicted LoF evasion or potential artifacts in 27.3% (304/1,113) of variants. The major reasons were location in the last exon, in a homopolymer repeat, in a low proportion expressed across transcripts (pext) scored region, or the presence of cryptic in-frame splice rescues. Variants predicted to evade LoF or to be potential artifacts were enriched for ClinVar benign variants. PVS1 was downgraded in 99.4% (162/163) of pLoF variants predicted as likely not LoF/not LoF, with 17.2% (28/163) downgraded as a result of our framework, adding to previous guidelines. Variant pathogenicity was affected (mostly from likely pathogenic to VUS) in 20 (71.4%) of these 28 variants. This framework guides assessment of pLoF variants beyond standard annotation pipelines and substantially reduces false positive rates, which is key to ensure accurate LoF variant prediction in both a research and clinical setting.