A supervised learning method for classifying methylation disorders.

BACKGROUND: DNA methylation is one of the most stable and well-characterized epigenetic alterations in humans. Accordingly, it has already found clinical utility as a molecular biomarker in a variety of disease contexts. Existing methods for clinical diagnosis of methylation-related disorders focus on outlier detection in a small number of CpG sites using standardized cutoffs which differentiate healthy from abnormal methylation levels. The standardized cutoff values used in these methods do not take into account methylation patterns which are known to differ between the sexes and with age. RESULTS: Here we profile genome-wide DNA methylation from blood samples drawn from within a cohort composed of healthy controls of different age and sex alongside patients with Prader-Willi syndrome (PWS), Beckwith-Wiedemann syndrome, Fragile-X syndrome, Angelman syndrome, and Silver-Russell syndrome. We propose a Generalized Additive Model to perform age and sex adjusted outlier analysis of around 700,000 CpG sites throughout the human genome. Utilizing z-scores among the cohort for each site, we deployed an ensemble based machine learning pipeline and achieved a combined prediction accuracy of 0.96 (Binomial 95% Confidence Interval 0.868[Formula: see text]0.995). CONCLUSION: We demonstrate a method for age and sex adjusted outlier detection of differentially methylated loci based on a large cohort of healthy individuals. We present a custom machine learning pipeline utilizing this outlier analysis to classify samples for potential methylation associated congenital disorders. These methods are able to achieve high accuracy when used with machine learning methods to classify abnormal methylation patterns.

The Importance of Mitochondrial Disease Testing in Young Adults With New Onset Sensorineural Hearing Loss.

OBJECTIVES: Sensorineural hearing loss (SNHL) occurs commonly as part of mitochondriopathies and varies in severity and onset. In this study, we characterized hearing with specific consideration for hearing loss as a potential early indicator of mitochondrial disease (MD). We hypothesize that genetic testing at the earliest detection of SNHL may lead to an earlier MD diagnosis. DESIGN: We reviewed the clinical and audiometric data of 49 patients undergoing genetic testing for MD. RESULTS: One-third of individuals with molecularly confirmed MD presented with SNHL. On average, patients had hearing loss at least 10 years before genetic testing. The collective audiometric profile includes mild to moderate SNHL at lower frequencies and moderate SNHL at 2 kHz and higher frequencies. CONCLUSIONS: This study suggests that screening for SNHL could be an early indicator of MD. We propose that the audiometric profile for those with a MD diagnosis may have clinical triage utility.

εγ-Thalassemia, a New Hemoglobinopathy Category.

BACKGROUND: Large ß-globin gene cluster deletions (hereditary persistence of fetal hemoglobin [Hb] or ß-, δß-, γδß-, and ϵγδß-thalassemia), are associated with widely disparate phenotypes, including variable degrees of microcytic anemia and Hb F levels. When present, increased Hb A2 is used as a surrogate marker for ß-thalassemia. Notably, ϵγδß-thalassemias lack the essential regulatory locus control region (LCR) and cause severe transient perinatal anemia but normal newborn screen (NBS) results and Hb A2 levels. Herein, we report a novel deletion of the ϵ, Aγ, Gγ, and ψß loci with intact LCR, δ-, and ß-regions in 2 women and newborn twins. METHODS: Capillary electrophoresis (CE), high-performance liquid chromatography (HPLC), DNA sequencing, multiplex ligation-dependent probe amplification (MLPA), gap-polymerase chain reaction (gap-PCR), and long-read sequencing (LRS) were performed. RESULTS: NBS showed an Hb A > Hb F pattern for both twins. At 20 months, Hb A2 was increased similarly to that in the mother and an unrelated woman. Unexplained microcytosis was absent and the twins lacked severe neonatal anemia. MLPA, LRS, and gap-PCR confirmed a 32 599 base pair deletion of ϵ (HBE1) through ψß (HBBP1) loci. CONCLUSIONS: This deletion represents a hemoglobinopathy category with a distinct phenotype that has not been previously described, an ϵγ-thalassemia. Both the NBS Hb A > F pattern and the subsequent increased Hb A2 without microcytosis are unusual. A similar deletion should be considered when this pattern is encountered and appropriate test methods selected for detection. Knowledge of the clinical impact of this new category will improve genetic counselling, with distinction from the severe transient anemia associated with ϵγδß-thalassemia.

Confirmation of Insertion, Deletion, and Deletion-Insertion Variants Detected by Next-Generation Sequencing.

BACKGROUND: Despite clinically demonstrated accuracy in next generation sequencing (NGS) data, many clinical laboratories continue to confirm variants with Sanger sequencing, which increases cost of testing and turnaround time. Several studies have assessed the accuracy of NGS in detecting single nucleotide variants; however, less has been reported about insertion, deletion, and deletion-insertion variants (indels). METHODS: We performed a retrospective analysis from 2015-2022 of indel results from a subset of NGS targeted gene panel tests offered through the Mayo Clinic Genomics Laboratories. We compared results from NGS and Sanger sequencing of indels observed in clinical runs and during the intra-assay validation of the tests. RESULTS: Results demonstrated 100% concordance between NGS and Sanger sequencing for over 490 indels (217 unique), ranging in size from 1 to 68 basepairs (bp). The majority of indels were deletions (77%) and 1 to 5 bp in length (90%). Variant frequencies ranged from 11.4% to 67.4% and 85.1% to 100% for heterozygous and homozygous variants, respectively, with a median depth of coverage of 2562×. A subset of indels (7%) were located in complex regions of the genome, and these were accurately detected by NGS. We also demonstrated 100% reproducibility of indel detection (n = 179) during intra-assay validation. CONCLUSIONS: Together this data demonstrates that reportable indel variants up to 68 bp can be accurately assessed using NGS, even when they occur in complex regions. Depending on the complexity of the region or variant, Sanger sequence confirmation of indels is usually not necessary if the variants meet appropriate coverage and allele frequency thresholds.

Uniparental disomy of multiple chromosomes in two cases with a complex phenotype.

Uniparental disomy (UPD) is the inheritance of both chromosomal homologs from one parent. Depending on the chromosome involved and the parental origin, UPD may result in phenotypic abnormalities due to aberrant methylation patterns or unmasking recessive conditions in isodisomic regions. UPD primarily originates from somatic rescue of a single meiotically-derived aneuploidy, most commonly a trisomy. Double UPD is exceedingly rare and triple UPD has not been previously described. Here, we report two unrelated clinical cases with UPD of multiple chromosomes; an 8-month-old male with maternal isodisomy of chromosome 7 and paternal isodisomy of chromosome 9, and a 4-week-old female with mixed paternal UPD for chromosomes 4, 10, and 14. These cases also demonstrate that although extremely rare, the detection of AOH on two or more chromosomes may warrant additional clinical and laboratory investigation such as methylation and STR marker analysis, especially when involving chromosomes known to be associated with imprinting disorders.

RINT1 Bi-allelic Variations Cause Infantile-Onset Recurrent Acute Liver Failure and Skeletal Abnormalities.

Pediatric acute liver failure (ALF) is life threatening with genetic, immunologic, and environmental etiologies. Approximately half of all cases remain unexplained. Recurrent ALF (RALF) in infants describes repeated episodes of severe liver injury with recovery of hepatic function between crises. We describe bi-allelic RINT1 alterations as the cause of a multisystem disorder including RALF and skeletal abnormalities. Three unrelated individuals with RALF onset ≤3 years of age have splice alterations at the same position (c.1333+1G>A or G>T) in trans with a missense (p.Ala368Thr or p.Leu370Pro) or in-frame deletion (p.Val618_Lys619del) in RINT1. ALF episodes are concomitant with fever/infection and not all individuals have complete normalization of liver function testing between episodes. Liver biopsies revealed nonspecific liver damage including fibrosis, steatosis, or mild increases in Kupffer cells. Skeletal imaging revealed abnormalities affecting the vertebrae and pelvis. Dermal fibroblasts showed splice-variant mediated skipping of exon 9 leading to an out-of-frame product and nonsense-mediated transcript decay. Fibroblasts also revealed decreased RINT1 protein, abnormal Golgi morphology, and impaired autophagic flux compared to control. RINT1 interacts with NBAS, recently implicated in RALF, and UVRAG, to facilitate Golgi-to-ER retrograde vesicle transport. During nutrient depletion or infection, Golgi-to-ER transport is suppressed and autophagy is promoted through UVRAG regulation by mTOR. Aberrant autophagy has been associated with the development of similar skeletal abnormalities and also with liver disease, suggesting that disruption of these RINT1 functions may explain the liver and skeletal findings. Clarifying the pathomechanism underlying this gene-disease relationship may inform therapeutic opportunities.

Clinical characterization of primary hyperoxaluria type 3 in comparison with types 1 and 2.

BACKGROUND: Primary hyperoxaluria (PH) type 3 (PH3) is caused by mutations in the hydroxy-oxo-glutarate aldolase 1 gene. PH3 patients often present with recurrent urinary stone disease in the first decade of life, but prior reports suggested PH3 may have a milder phenotype in adults. This study characterized clinical manifestations of PH3 across the decades of life in comparison with PH1 and PH2. METHODS: Clinical information was obtained from the Rare Kidney Stone Consortium PH Registry (PH1, n = 384; PH2, n = 51; PH3, n = 62). RESULTS: PH3 patients presented with symptoms at a median of 2.7 years old compared with PH1 (4.9 years) and PH2 (5.7 years) (P = 0.14). Nephrocalcinosis was present at diagnosis in 4 (7%) PH3 patients, while 55 (89%) had stones. Median urine oxalate excretion was lowest in PH3 patients compared with PH1 and PH2 (1.1 versus 1.6 and 1.5 mmol/day/1.73 m2, respectively, P < 0.001) while urine calcium was highest in PH3 (112 versus 51 and 98 mg/day/1.73 m2 in PH1 and PH2, respectively, P < 0.001). Stone events per decade of life were similar across the age span and the three PH types. At 40 years of age, 97% of PH3 patients had not progressed to end-stage kidney disease compared with 36% PH1 and 66% PH2 patients. CONCLUSIONS: Patients with all forms of PH experience lifelong stone events, often beginning in childhood. Kidney failure is common in PH1 but rare in PH3. Longer-term follow-up of larger cohorts will be important for a more complete understanding of the PH3 phenotype.

WDR37 syndrome: identification of a distinct new cluster of disease-associated variants and functional analyses of mutant proteins.

Missense variants located in the N-terminal region of WDR37 were recently identified to cause a multisystemic syndrome affecting neurological, ocular, gastrointestinal, genitourinary, and cardiac development. WDR37 encodes a WD40 repeat-containing protein of unknown function. We identified three novel WDR37 variants, two likely pathogenic de novo alleles and one inherited variant of uncertain significance, in individuals with phenotypes overlapping those previously reported but clustering in a different region of the protein. The novel alleles are C-terminal to the prior variants and located either within the second WD40 motif (c.659A>G p.(Asp220Gly)) or in a disordered protein region connecting the second and third WD40 motifs (c.778G>A p.(Asp260Asn) and c.770C>A p.(Pro257His)). The three novel mutants showed normal cellular localization but lower expression levels in comparison to wild-type WDR37. To investigate the normal interactions of WDR37, we performed co-immunoprecipitation and yeast two-hybrid assays. This revealed the ability of WDR37 to form homodimers and to strongly bind PACS1 and PACS2 phosphofurin acidic cluster sorting proteins; immunocytochemistry confirmed colocalization of WDR37 with PACS1 and PACS2 in human cells. Next, we analyzed previously reported and novel mutants for their ability to dimerize with wild-type WDR37 and bind PACS proteins. Interaction with wild-type WDR37 was not affected for any variant; however, one novel mutant, p.(Asp220Gly), lost its ability to bind PACS1 and PACS2. In summary, this study presents a novel region of WDR37 involved in human disease, identifies PACS1 and PACS2 as major binding partners of WDR37 and provides insight into the functional effects of various WDR37 variants.

Impact of integrated translational research on clinical exome sequencing.

PURPOSE: Exome sequencing often identifies pathogenic genetic variants in patients with undiagnosed diseases. Nevertheless, frequent findings of variants of uncertain significance necessitate additional efforts to establish causality before reaching a conclusive diagnosis. To provide comprehensive genomic testing to patients with undiagnosed disease, we established an Individualized Medicine Clinic, which offered clinical exome testing and included a Translational Omics Program (TOP) that provided variant curation, research activities, or research exome sequencing. METHODS: From 2012 to 2018, 1101 unselected patients with undiagnosed diseases received exome testing. Outcomes were reviewed to assess impact of the TOP and patient characteristics on diagnostic rates through descriptive and multivariate analyses. RESULTS: The overall diagnostic yield was 24.9% (274 of 1101 patients), with 174 (15.8% of 1101) diagnosed on the basis of clinical exome sequencing alone. Four hundred twenty-three patients with nondiagnostic or without access to clinical exome sequencing were evaluated by the TOP, with 100 (9% of 1101) patients receiving a diagnosis, accounting for 36.5% of the diagnostic yield. The identification of a genetic diagnosis was influenced by the age at time of testing and the disease phenotype of the patient. CONCLUSION: Integration of translational research activities into clinical practice of a tertiary medical center can significantly increase the diagnostic yield of patients with undiagnosed disease.

Mutations in the V-ATPase Assembly Factor VMA21 Cause a Congenital Disorder of Glycosylation With Autophagic Liver Disease.

BACKGROUND AND AIMS: Vacuolar H+-ATP complex (V-ATPase) is a multisubunit protein complex required for acidification of intracellular compartments. At least five different factors are known to be essential for its assembly in the endoplasmic reticulum (ER). Genetic defects in four of these V-ATPase assembly factors show overlapping clinical features, including steatotic liver disease and mild hypercholesterolemia. An exception is the assembly factor vacuolar ATPase assembly integral membrane protein (VMA21), whose X-linked mutations lead to autophagic myopathy. APPROACH AND RESULTS: Here, we report pathogenic variants in VMA21 in male patients with abnormal protein glycosylation that result in mild cholestasis, chronic elevation of aminotransferases, elevation of (low-density lipoprotein) cholesterol and steatosis in hepatocytes. We also show that the VMA21 variants lead to V-ATPase misassembly and dysfunction. As a consequence, lysosomal acidification and degradation of phagocytosed materials are impaired, causing lipid droplet (LD) accumulation in autolysosomes. Moreover, VMA21 deficiency triggers ER stress and sequestration of unesterified cholesterol in lysosomes, thereby activating the sterol response element-binding protein-mediated cholesterol synthesis pathways. CONCLUSIONS: Together, our data suggest that impaired lipophagy, ER stress, and increased cholesterol synthesis lead to LD accumulation and hepatic steatosis. V-ATPase assembly defects are thus a form of hereditary liver disease with implications for the pathogenesis of nonalcoholic fatty liver disease.

CSNK2B: A broad spectrum of neurodevelopmental disability and epilepsy severity.

CSNK2B has recently been implicated as a disease gene for neurodevelopmental disability (NDD) and epilepsy. Information about developmental outcomes has been limited by the young age and short follow-up for many of the previously reported cases, and further delineation of the spectrum of associated phenotypes is needed. We present 25 new patients with variants in CSNK2B and refine the associated NDD and epilepsy phenotypes. CSNK2B variants were identified by research or clinical exome sequencing, and investigators from different centers were connected via GeneMatcher. Most individuals had developmental delay and generalized epilepsy with onset in the first 2 years. However, we found a broad spectrum of phenotypic severity, ranging from early normal development with pharmacoresponsive seizures to profound intellectual disability with intractable epilepsy and recurrent refractory status epilepticus. These findings suggest that CSNK2B should be considered in the diagnostic evaluation of patients with a broad range of NDD with treatable or intractable seizures.

Integrating glycomics and genomics uncovers SLC10A7 as essential factor for bone mineralization by regulating post-Golgi protein transport and glycosylation.

Genomics methodologies have significantly improved elucidation of Mendelian disorders. The combination with high-throughput functional-omics technologies potentiates the identification and confirmation of causative genetic variants, especially in singleton families of recessive inheritance. In a cohort of 99 individuals with abnormal Golgi glycosylation, 47 of which being unsolved, glycomics profiling was performed of total plasma glycoproteins. Combination with whole-exome sequencing in 31 cases revealed a known genetic defect in 15 individuals. To identify additional genetic factors, hierarchical clustering of the plasma glycomics data was done, which indicated a subgroup of four patients that shared a unique glycomics signature of hybrid type N-glycans. In two siblings, compound heterozygous mutations were found in SLC10A7, a gene of unknown function in human. These included a missense mutation that disrupted transmembrane domain 4 and a mutation in a splice acceptor site resulting in skipping of exon 9. The two other individuals showed a complete loss of SLC10A7 mRNA. The patients' phenotype consisted of amelogenesis imperfecta, skeletal dysplasia, and decreased bone mineral density compatible with osteoporosis. The patients' phenotype was mirrored in SLC10A7 deficient zebrafish. Furthermore, alizarin red staining of calcium deposits in zebrafish morphants showed a strong reduction in bone mineralization. Cell biology studies in fibroblasts of affected individuals showed intracellular mislocalization of glycoproteins and a defect in post-Golgi transport of glycoproteins to the cell membrane. In contrast to yeast, human SLC10A7 localized to the Golgi. Our combined data indicate an important role for SLC10A7 in bone mineralization and transport of glycoproteins to the extracellular matrix.

Congenital ichthyosis in Prader-Willi syndrome associated with maternal chromosome 15 uniparental disomy: Case report and review of autosomal recessive conditions unmasked by UPD.

Prader-Willi syndrome (PWS) is a prototypic genetic condition related to imprinting. Causative mechanisms include paternal 15q11-q13 deletion, maternal chromosome 15 uniparental disomy (UPD15), Prader-Willi Syndrome/Angelman Syndrome (PWS/AS) critical region imprinting defects, and complex chromosomal rearrangements. Maternal UPD15-related PWS poses risks of concomitant autosomal recessive (AR) disorders when the mother carries a pathogenic variant in one of the genes on chromosome 15 associated with autosomal recessive inherited disease. Co-occurrence of autosomal recessive conditions in the setting of UPD leads to increased complexity of the clinical phenotype, and may delay the diagnosis of PWS. We report a patient with PWS and associated congenital ichthyosis due to maternal UPD15, and a homozygous novel pathogenic variant in ceramide synthase 3 (CERS3). We also review the literature of associated disorders reported in the setting of maternal UPD15-related PWS and provide a summary of the previously described CERS3 variants. This represents the second case of autosomal recessive congenital ichthyosis (ARCI) in the setting of PWS and UPD15. There needs to be a high index of suspicion of this genetic mechanism when there is unexpected phenotype or evolution of the clinical course in a patient with PWS.

De novo DDX3X missense variants in males appear viable and contribute to syndromic intellectual disability.

DDX3X (Xp11.4) encodes a DEAD-box RNA helicase that escapes X chromosome inactivation. Pathogenic variants in DDX3X have been shown to cause X-linked intellectual disability (ID) (MRX102, MIM: 300958). The phenotypes associated with DDX3X variants are heterogeneous and include brain and behavioral abnormalities, microcephaly, hypotonia, and movement disorders and/or spasticity. The majority of DDX3X variants described are de novo mutations in females with ID. In contrast, most male DDX3X variants are inherited from an unaffected mother, with one documented exception being a recently identified de novo splice site variant. It has been suggested, therefore, that DDX3X exerts its effects through haploinsufficiency in females, and that affected males carry hypomorphic alleles that retain partial function. Given the lack of male de novo DDX3X variants, loss-of-function variants in this gene are suspected to be male lethal. Through whole-exome sequencing, we identified three unrelated males with hemizygous missense DDX3X variants and ID. All three variants were confirmed by Sanger sequencing, with two established as de novo. In silico analyses were supportive of pathogenicity. We report the male phenotypes and compare them to phenotypes observed in previously reported male and female patients. In conclusion, we propose that de novo DDX3X variants are not necessarily male lethal and should be considered as a cause of syndromic ID in both males and females.

Expert specification of the ACMG/AMP variant interpretation guidelines for genetic hearing loss.

Due to the high genetic heterogeneity of hearing loss (HL), current clinical testing includes sequencing large numbers of genes, which often yields a significant number of novel variants. Therefore, the standardization of variant interpretation is crucial to provide consistent and accurate diagnoses. The Hearing Loss Variant Curation Expert Panel was created within the Clinical Genome Resource to provide expert guidance for standardized genomic interpretation in the context of HL. As one of its major tasks, our Expert Panel has adapted the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines for the interpretation of sequence variants in HL genes. Here, we provide a comprehensive illustration of the newly specified ACMG/AMP HL rules. Three rules remained unchanged, four rules were removed, and the remaining 21 rules were specified. These rules were further validated and refined using a pilot set of 51 variants assessed by curators and disease experts. Of the 51 variants evaluated in the pilot, 37% (19/51) changed category based upon application of the expert panel specified rules and/or aggregation of evidence across laboratories. These HL-specific ACMG/AMP rules will help standardize variant interpretation, ultimately leading to better care for individuals with HL.

Developmental delay and failure to thrive associated with a loss-of-function variant in WHSC1 (NSD2).

Wolf-Hirschhorn syndrome (WHS) is a microdeletion syndrome characterized by distinctive facial features consisting of "Greek warrior helmet" appearance, prenatal and postnatal growth deficiency, developmental disability, and seizures. This disorder is caused by heterozygous deletions on chromosome 4p16.3 often identified by cytogenetic techniques. Many groups have attempted to identify the critical region within this deletion to establish which genes are responsible for WHS. Herein, clinical whole exome sequencing (WES) was performed on a child with developmental delays, mild facial dysmorphisms, short stature, failure to thrive, and microcephaly, and revealed a de novo frameshift variant, c.1676_1679del (p.Arg559Tfs*38), in WHSC1 (NSD2). While WHSC1 falls within the WHS critical region, individuals with only disruption of this gene have only recently been described in the literature. Loss-of-function de novo variations in WHSC1 were identified in large developmental delay, autism, diagnostic, and congenital cardiac cohorts, as well as recent case reports, suggesting that de novo loss-of-function WHSC1 variants may be related to disease. These findings, along with our patient suggest that loss-of-function variation in WHSC1 may lead to a mild form of Wolf-Hirschhorn syndrome, and also may suggest that the developmental delays, facial dysmorphisms, and short stature seen in WHS may be due to disruption of WHSC1 gene.

Diagnostic Tools for Inborn Errors of Human Immunity (Primary Immunodeficiencies and Immune Dysregulatory Diseases).

PURPOSE OF REVIEW: The purpose of this review is to provide an overview of diagnostic testing in primary immunodeficiency and immune dysregulatory disorders (PIDDs), particularly focusing on flow cytometry and genetic techniques, utilizing specific examples of PIDDs. RECENT FINDINGS: Flow cytometry remains a vital tool in the diagnosis and monitoring of immunological diseases. Its utility ranges from cellular analysis and specific protein quantitation to functional assays and signaling pathway analysis. Mass cytometry combines flow cytometry and mass spectrometry to dramatically increase the throughput of multivariate single-cell analysis. Next-generation sequencing in combination with other molecular techniques and processing algorithms has become more widely available and identified the diverse and heterogeneous genetic underpinnings of these disorders. As the spectrum of disease is further clarified by increasing immunological, genetic, and epigenetic knowledge, the careful application of these diagnostic tools and bioinformatics will assist not only in our understanding of these complex disorders, but also enable the implementation of personalized therapeutic approaches for disease management.