Diagnostic utility and reporting recommendations for clinical DNA methylation episignature testing in genetically undiagnosed rare diseases.

PURPOSE: This study aims to assess the diagnostic utility and provide reporting recommendations for clinical DNA methylation episignature testing based on the cohort of patients tested through the EpiSign Clinical Testing Network. METHODS: The EpiSign assay utilized unsupervised clustering techniques and a support vector machine-based classification algorithm to compare each patient's genome-wide DNA methylation profile with the EpiSign Knowledge Database, yielding the result that was reported. An international working group, representing distinct EpiSign Clinical Testing Network health jurisdictions, collaborated to establish recommendations for interpretation and reporting of episignature testing. RESULTS: Among 2399 cases analyzed, 1667 cases underwent a comprehensive screen of validated episignatures, imprinting, and promoter regions, resulting in 18.7% (312/1667) positive reports. The remaining 732 referrals underwent targeted episignature analysis for assessment of sequence or copy-number variants (CNVs) of uncertain significance or for assessment of clinical diagnoses without confirmed molecular findings, and 32.4% (237/732) were positive. Cases with detailed clinical information were highlighted to describe various utility scenarios for episignature testing. CONCLUSION: Clinical DNA methylation testing including episignatures, imprinting, and promoter analysis provided by an integrated network of clinical laboratories enables test standardization and demonstrates significant diagnostic yield and clinical utility beyond DNA sequence analysis in rare diseases.

X-Linked intellectual disability update 2022.

Genes that are involved in the transcription process, mitochondrial function, glycoprotein metabolism, and ubiquitination dominate the list of 21 new genes associated with X-linked intellectual disability since the last update in 2017. The new genes were identified by sequencing of candidate genes (2), the entire X-chromosome (2), the whole exome (15), or the whole genome (2). With these additions, 42 (21%) of the 199 named XLID syndromes and 27 (25%) of the 108 numbered nonsyndromic XLID families remain to be resolved at the molecular level. Although the pace of discovery of new XLID genes has slowed during the past 5 years, the density of genes on the X chromosome that cause intellectual disability still appears to be twice the density of intellectual disability genes on the autosomes.

Recommendations by the ClinGen Rett/Angelman-like expert panel for gene-specific variant interpretation methods.

The genes MECP2, CDKL5, FOXG1, UBE3A, SLC9A6, and TCF4 present unique challenges for current ACMG/AMP variant interpretation guidelines. To address those challenges, the Rett and Angelman-like Disorders Variant Curation Expert Panel (Rett/AS VCEP) drafted gene-specific modifications. A pilot study was conducted to test the clarity and accuracy of using the customized variant interpretation criteria. Multiple curators obtained the same interpretation for 78 out of the 87 variants (~90%), indicating appropriate usage of the modified guidelines the majority of times by all the curators. The classification of 13 variants changed using these criteria specifications compared to when the variants were originally curated and as present in ClinVar. Many of these changes were due to internal data shared from laboratory members however some changes were because of changes in strength of criteria. There were no two-step classification changes and only 1 clinically relevant change (Likely pathogenic to VUS). The Rett/AS VCEP hopes that these gene-specific variant curation rules and the assertions provided help clinicians, clinical laboratories, and others interpret variants in these genes but also other fully penetrant, early-onset genes associated with rare disorders.

Functional correlation of genome-wide DNA methylation profiles in genetic neurodevelopmental disorders.

An expanding range of genetic syndromes are characterized by genome-wide disruptions in DNA methylation profiles referred to as episignatures. Episignatures are distinct, highly sensitive, and specific biomarkers that have recently been applied in clinical diagnosis of genetic syndromes. Episignatures are contained within the broader disorder-specific genome-wide DNA methylation changes, which can share significant overlap among different conditions. In this study, we performed functional genomic assessment and comparison of disorder-specific and overlapping genome-wide DNA methylation changes related to 65 genetic syndromes with previously described episignatures. We demonstrate evidence of disorder-specific and recurring genome-wide differentially methylated probes (DMPs) and regions (DMRs). The overall distribution of DMPs and DMRs across the majority of the neurodevelopmental genetic syndromes analyzed showed substantial enrichment in gene promoters and CpG islands, and under-representation of the more variable intergenic regions. Analysis showed significant enrichment of the DMPs and DMRs in gene pathways and processes related to neurodevelopment, including neurogenesis, synaptic signaling and synaptic transmission. This study expands beyond the diagnostic utility of DNA methylation episignatures by demonstrating correlation between the function of the mutated genes and the consequent genomic DNA methylation profiles as a key functional element in the molecular etiology of genetic neurodevelopmental disorders.

Mosaicism of common pathogenic MECP2 variants identified in two males with a clinical diagnosis of Rett syndrome.

Rett (RTT) syndrome, a neurodevelopmental disorder caused by pathogenic variation in the MECP2 gene, is characterized by developmental regression, loss of purposeful hand movements, stereotypic hand movements, abnormal gait, and loss of spoken language. Due to the X-linked inheritance pattern, RTT is typically limited to females. Recent studies revealed somatic mosaicism in MECP2 in male patients with RTT-like phenotypes. While detecting mosaic variation using Sanger sequencing is theoretically possible for mosaicism over ~15%-20%, several variables, including efficiency of PCR, background noise, and/or human error, contribute to a low detection rate using this technology. Mosaic variants in two males were detected by next generation sequencing (NGS; Case 1) and by Sanger re-sequencing (Case 2). Both had targeted digital PCR (dPCR) to confirm the variants. In this report, we present two males with classic RTT syndrome in whom we identified pathogenic variation in the MECP2 gene in the mosaic state (c.730C > T (p.Gln244*) in Patient 1 and c.397C > T (p.Arg133Cys) in Patient 2). In addition, estimates and measures of mosaic variant fraction were surprisingly similar between Sanger sequencing, NGS, and dPCR. The mosaic state of these variants contributed to a lengthy diagnostic odyssey for these patients. While NGS and even Sanger sequencing may be viable methods of detecting mosaic variation in DNA or RNA samples, applying targeted dPCR to supplement these sequencing technologies would provide confirmation of somatic mosaicism and mosaic fraction.

Genetics of agenesis/hypoplasia of the uterus and vagina: narrowing down the number of candidate genes for Mayer-Rokitansky-Küster-Hauser Syndrome.

PURPOSE: Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome consists of congenital absence of the uterus and vagina and is often associated with renal, skeletal, cardiac, and auditory defects. The genetic basis is largely unknown except for rare variants in several genes. Many candidate genes have been suggested by mouse models and human studies. The purpose of this study was to narrow down the number of candidate genes. METHODS: Whole exome sequencing was performed on 111 unrelated individuals with MRKH; variant analysis focused on 72 genes suggested by mouse models, human studies of physiological candidates, or located near translocation breakpoints in t(3;16). Candidate variants (CV) predicted to be deleterious were confirmed by Sanger sequencing. RESULTS: Sanger sequencing verified 54 heterozygous CV from genes identified through mouse (13 CV in 6 genes), human (22 CV in seven genes), and translocation breakpoint (19 CV in 11 genes) studies. Twelve patients had ≥ 2 CVs, including four patients with two variants in the same gene. One likely digenic combination of LAMC1 and MMP14 was identified. CONCLUSION: We narrowed 72 candidate genes to 10 genes that appear more likely implicated. These candidate genes will require further investigation to elucidate their role in the development of MRKH.

Clinical epigenomics: genome-wide DNA methylation analysis for the diagnosis of Mendelian disorders.

PURPOSE: We describe the clinical implementation of genome-wide DNA methylation analysis in rare disorders across the EpiSign diagnostic laboratory network and the assessment of results and clinical impact in the first subjects tested. METHODS: We outline the logistics and data flow between an integrated network of clinical diagnostics laboratories in Europe, the United States, and Canada. We describe the clinical validation of EpiSign using 211 specimens and assess the test performance and diagnostic yield in the first 207 subjects tested involving two patient subgroups: the targeted cohort (subjects with previous ambiguous/inconclusive genetic findings including genetic variants of unknown clinical significance) and the screening cohort (subjects with clinical findings consistent with hereditary neurodevelopmental syndromes and no previous conclusive genetic findings). RESULTS: Among the 207 subjects tested, 57 (27.6%) were positive for a diagnostic episignature including 48/136 (35.3%) in the targeted cohort and 8/71 (11.3%) in the screening cohort, with 4/207 (1.9%) remaining inconclusive after EpiSign analysis. CONCLUSION: This study describes the implementation of diagnostic clinical genomic DNA methylation testing in patients with rare disorders. It provides strong evidence of clinical utility of EpiSign analysis, including the ability to provide conclusive findings in the majority of subjects tested.

Expanding the clinical and metabolic phenotype of DPM2 deficient congenital disorders of glycosylation.

Pathogenic alterations in the DPM2 gene have been previously described in patients with hypotonia, progressive muscle weakness, absent psychomotor development, intractable seizures, and early death. We identified biallelic DPM2 variants in a 23-year-old male with truncal hypotonia, hypertonicity, congenital heart defects, intellectual disability, and generalized muscle wasting. His clinical presentation was much less severe than that of the three previously described patients. This is the second report on this ultra-rare disorder. Here we review the characteristics of previously reported individuals with a defect in the DPM complex while expanding the clinical phenotype of DPM2-Congenital Disorders of Glycosylation. In addition, we offer further insights into the pathomechanism of DPM2-CDG disorder by introducing glycomics and lipidomics analysis.

Xq22 deletions and correlation with distinct neurological disease traits in females: Further evidence for a contiguous gene syndrome.

Xq22 deletions that encompass PLP1 (Xq22-PLP1-DEL) are notable for variable expressivity of neurological disease traits in females ranging from a mild late-onset form of spastic paraplegia type 2 (MIM# 312920), sometimes associated with skewed X-inactivation, to an early-onset neurological disease trait (EONDT) of severe developmental delay, intellectual disability, and behavioral abnormalities. Size and gene content of Xq22-PLP1-DEL vary and were proposed as potential molecular etiologies underlying variable expressivity in carrier females where two smallest regions of overlap (SROs) were suggested to influence disease. We ascertained a cohort of eight unrelated patients harboring Xq22-PLP1-DEL and performed high-density array comparative genomic hybridization and breakpoint-junction sequencing. Molecular characterization of Xq22-PLP1-DEL from 17 cases (eight herein and nine published) revealed an overrepresentation of breakpoints that reside within repeats (11/17, ~65%) and the clustering of ~47% of proximal breakpoints in a genomic instability hotspot with characteristic non-B DNA density. These findings implicate a potential role for genomic architecture in stimulating the formation of Xq22-PLP1-DEL. The correlation of Xq22-PLP1-DEL gene content with neurological disease trait in female cases enabled refinement of the associated SROs to a single genomic interval containing six genes. Our data support the hypothesis that genes contiguous to PLP1 contribute to EONDT.

Whole-Genome and Segmental Homozygosity Confirm Errors in Meiosis as Etiology of Struma Ovarii.

Strumae ovarii are neoplasms composed of normal-appearing thyroid tissue that occur within the ovary and rarely spread to extraovarian sites. A unique case of struma ovarii with widespread dissemination detected 48 years after removal of a pelvic dermoid provided the opportunity to reexamine the molecular nature of this form of neoplasm. One tumor, from the heart, consisting of benign thyroid tissue was found to have whole-genome homozygosity. Another tumor from the right mandible composed of malignant-appearing thyroid tissue showed whole-genome homozygosity and a deletion of 7p, presumably the second hit that transformed it into a cancerous tumor. Specimens from 2 other cases of extraovarian struma confined to the abdomen and 8 of 9 cases of intraovarian struma showed genome-wide segmental homozygosity. These findings confirm errors in meiosis as the origin of struma ovarii. The histological and molecular findings further demonstrate that even when outside the ovary, strumae ovarii can behave nonaggressively until they receive a second hit, thereafter behaving like cancer.

Schimke XLID syndrome results from a deletion in BCAP31.

A family with three affected males and a second family with a single affected male with intellectual disability, microcephaly, ophthalmoplegia, deafness, and Involuntary limb movements were reported by Schimke and Associates in 1984. The affected males with Schimke X-linked intellectual disability (XLID) syndrome (OMIM# 312840) had a similar facial appearance with deep-set eyes, downslanting palpebral fissures, hypotelorism, narrow nose and alae nasi, cupped ears and spacing of the teeth. Two mothers had mild hearing loss but no other manifestations of the disorder. The authors considered the disorder to be distinctive and likely X-linked. Whole genome sequencing in the single affected male available and the three carrier females from one of the families with Schimke XLID syndrome identified a 2 bp deletion in the BCAP31 gene. During the past decade, pathogenic alterations of the BCAP31 gene have been associated with deafness, dystonia, and central hypomyelination, an XLID condition given the eponym DDCH syndrome. A comparison of clinical findings in Schimke XLID syndrome and DDCH syndrome shows them to be the same clinical entity. The BCAP31 protein functions in endoplasmic reticulum-associated degradation to promote ubiquitination and destruction of misfolded proteins.

The lysosomal storage disorders mucolipidosis type II, type III alpha/beta, and type III gamma: Update on GNPTAB and GNPTG mutations.

Mutations in the GNPTAB and GNPTG genes cause mucolipidosis (ML) type II, type III alpha/beta, and type III gamma, which are autosomal recessively inherited lysosomal storage disorders. GNPTAB and GNPTG encode the α/ß-precursor and the γ-subunit of N-acetylglucosamine (GlcNAc)-1-phosphotransferase, respectively, the key enzyme for the generation of mannose 6-phosphate targeting signals on lysosomal enzymes. Defective GlcNAc-1-phosphotransferase results in missorting of lysosomal enzymes and accumulation of non-degradable macromolecules in lysosomes, strongly impairing cellular function. MLII-affected patients have coarse facial features, cessation of statural growth and neuromotor development, severe skeletal abnormalities, organomegaly, and cardiorespiratory insufficiency leading to death in early childhood. MLIII alpha/beta and MLIII gamma are attenuated forms of the disease. Since the identification of the GNPTAB and GNPTG genes, 564 individuals affected by MLII or MLIII have been described in the literature. In this report, we provide an overview on 258 and 50 mutations in GNPTAB and GNPTG, respectively, including 58 novel GNPTAB and seven novel GNPTG variants. Comprehensive functional studies of GNPTAB missense mutations did not only gain insights into the composition and function of the GlcNAc-1-phosphotransferase, but also helped to define genotype-phenotype correlations to predict the clinical outcome in patients.

Three additional patients with EED-associated overgrowth: potential mutation hotspots identified?

Variants have been identified in the embryonic ectoderm development (EED) gene in seven patients with syndromic overgrowth similar to that observed in Weaver syndrome. Here, we present three additional patients with missense variants in the EED gene. All the missense variants reported to date (including the three presented here) have localized to one of seven WD40 domains of the EED protein, which are necessary for interaction with enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2). In addition, among the seven patients reported in the literature and the three new patients presented here, all of the reported pathogenic variants except one occurred at one of four amino acid residues in the EED protein. The recurrence of pathogenic variation at these loci suggests that these residues are functionally important (mutation hotspots). In silico modeling and calculations of the free energy changes resulting from these variants suggested that they not only destabilize the EED protein structure but also adversely affect interactions between EED, EZH2, and/or H3K27me3. These cases help demonstrate the mechanism(s) by which apparently deleterious variants in the EED gene might cause overgrowth and lend further support that amino acid residues in the WD40 domain region may be mutation hotspots.

Biallelic deletions of the Waardenburg II syndrome gene, SOX10, cause a recognizable arthrogryposis syndrome.

Random mating in the general population tends to limit the occurrence of homozygous and compound heterozygous forms of dominant hereditary disorders. Certain phenotypes, the most recognized being skeletal dysplasias associated with short stature, lead to cultural interaction and assortative mating. To this well-known example, may be added deafness which brings together individuals with a variety of deafness genotypes, some being dominant. Waardenburg syndrome is one such autosomal dominant disorder in which affected individuals may interact culturally because of deafness. Biallelic genetic alterations for two Waardenburg genes, PAX3 and MITF have been previously recognized. Herein, we report biallelic deletions in SOX10, a gene associated with Waardenburg syndromes type II and IV. The affected fetuses have a severe phenotype with a lack of fetal movement resulting in four-limb arthrogryposis and absence of palmar and plantar creases, white hair, dystopia canthorum, and in one case cleft palate and in the other a cardiac malformation.

Enrichment of mutations in chromatin regulators in people with Rett syndrome lacking mutations in MECP2.

PURPOSE: Rett syndrome (RTT) is a neurodevelopmental disorder caused primarily by de novo mutations in MECP2 and sometimes in CDKL5 and FOXG1. However, some RTT patients lack mutations in these genes. METHODS: Twenty-two RTT patients without apparent MECP2, CDKL5, and FOXG1 mutations were subjected to both whole-exome sequencing and single-nucleotide polymorphism array-based copy-number variant (CNV) analyses. RESULTS: Three patients had MECP2 mutations initially missed by clinical testing. Of the remaining 19, 17 (89.5%) had 29 other likely pathogenic intragenic mutations and/or CNVs (10 patients had 2 or more). Interestingly, 13 patients had mutations in a gene/region previously reported in other neurodevelopmental disorders (NDDs), thereby providing a potential diagnostic yield of 68.4%. These mutations were significantly enriched in chromatin regulators (corrected P = 0.0068) and moderately enriched in postsynaptic cell membrane molecules (corrected P = 0.076), implicating glutamate receptor signaling. CONCLUSION: The genetic etiology of RTT without MECP2, CDKL5, and FOXG1 mutations is heterogeneous, overlaps with other NDDs, and complicated by a high mutation burden. Dysregulation of chromatin structure and abnormal excitatory synaptic signaling may form two common pathological bases of RTT.Genet Med 19 1, 13-19.

Novel pathogenic variants in FOXP3 in fetuses with echogenic bowel and skin desquamation identified by ultrasound.

Immunodysregulation, Polyendocrinopathy, Enteropathy, X-linked (IPEX) syndrome is a rare, X-linked recessive disease that affects regulatory T cells (Tregs) resulting in diarrhea, enteropathy, eczema, and insulin-dependent diabetes mellitus. IPEX syndrome is caused by pathogenic alterations in FOXP3 located at Xp11.23. FOXP3 encodes a transcription factor that interacts with several partners, including NFAT and NF-κB, and is necessary for the proper cellular differentiation of Tregs. Although variable, the vast majority of IPEX syndrome patients have onset of disease during infancy with severe enteropathy. Only five families with prenatal presentation of IPEX syndrome have been reported. Here, we present two additional prenatal onset cases with novel inherited frameshift pathogenic variants in FOXP3 that generate premature stop codons. Ultrasound findings in the first patient identified echogenic bowel, echogenic debris, scalp edema, and hydrops. In the second patient, ultrasound findings included polyhydramnios with echogenic debris, prominent fluid-filled loops of bowel, and echogenic bowel. These cases further broaden the phenotypic spectrum of IPEX syndrome by describing previously unappreciated prenatal ultrasound findings associated with the disease.

New observation of sialuria prompts detection of liver tumor in previously reported patient.

UNLABELLED: Sialuria, a rare inborn error of metabolism, was diagnosed in a healthy 12-year-old boy through whole exome sequencing. The patient had experienced mild delays of speech and motor development, as well as persistent hepatomegaly. Identification of the 8th individual with this disorder, prompted follow-up of the mother-son pair of patients diagnosed over 15years ago. Hepatomegaly was confirmed in the now 19-year-old son, but in the 46-year-old mother a clinically silent liver tumor was detected by ultrasound and MRI. The tumor was characterized as an intrahepatic cholangiocarcinoma (IHCC) and DNA analysis of both tumor and normal liver tissue confirmed the original GNE mutation. As the maternal grandmother in the latter family died at age 49years of a liver tumor, a retrospective study of the remaining pathology slides was conducted and confirmed it to have been an IHCC as well. The overall observation generated the hypothesis that sialuria may predispose to development of this form of liver cancer. As proof of sialuria in the grandmother could not be obtained, an alternate cause of IHCC cannot be ruled out. In a series of 102 patients with IHCC, not a single instance was found with the allosteric site mutation in the GNE gene. This confirms that sialuria is rare even in a selected group of patients, but does not invalidate the concern that sialuria may be a risk factor for IHCC. SYNOPSIS: Sialuria is a rare inborn error of metabolism characterized by excessive synthesis and urinary excretion of free sialic acid with only minimal clinical morbidity in early childhood, but may be a risk factor for intrahepatic cholangiocarcinoma in adulthood.

A novel familial autosomal dominant mutation in ARID1B causing neurodevelopmental delays, short stature, and dysmorphic features.

Recent studies have identified mutations in the ARID1B gene responsible for neurodevelopmental delays, intellectual disability, growth delay, and dysmorphic features. ARID1B encodes a subunit of the BAF chromatin-remodeling complex, and mutations in multiple components of the BAF complex have been implicated as causes of Coffin-Siris syndrome, Nicolaides-Baraitser syndrome, and non-syndromic intellectual disability. The majority of documented pathogenic ARID1B mutations to date have arisen in a sporadic, de novo manner with no reports of inheritance of a pathogenic mutation from an affected parent. We describe here two patients (a 21-year-old female and her 21-month-old son) with a novel frameshift mutation in ARID1B inherited in an autosomal dominant fashion in the affected offspring. Both patients presented with neurodevelopmental delays, growth delay, and dysmorphic features including prominent nose with full nasal tip, long philtrum, and high-arched palate. Exome sequencing analysis in the female patient demonstrated a heterozygous deletion of nucleotide 1259 of the ARID1B gene (c.1259delA) resulting in a frameshift and creation of a premature stop codon. Further family testing by targeted Sanger sequencing confirmed that this arose as a de novo mutation in the mother and was passed on to her affected son. The clinical features of both patients are felt to be consistent with an ARID1B-related disorder. To our knowledge, this is the first report of a pathogenic mutation in ARID1B being passed from an affected parent to their offspring. © 2016 Wiley Periodicals, Inc.

Identification of a novel insertion mutation in FGFR3 that causes thanatophoric dysplasia type 1.

Thanatophoric dysplasia is a type of short-limbed neonatal dwarfism that is usually lethal in the perinatal period. It is characterized by short limbs, a narrow, bell-shaped thorax, macrocephaly with a prominent forehead, and flattened vertebral bodies. These malformations result from autosomal dominant mutations in the fibroblast growth factor receptor 3 (FGFR3) gene. In this report, we describe a novel FGFR3 insertion mutation in a fetus with shortened limbs, curved femurs, and a narrow thorax. The diagnosis of thanatophoric dysplasia type 1 was suspected clinically, and FGFR3 sequencing showed a c.742_743insTGT variant, which predicts p.R248delinsLC. In vivo studies in zebrafish demonstrated that this mutation resulted in the overexpression of zebrafish Fgfr3, leading to the over-activation of downstream signaling and dorsalized embryos. To date, no insertions or deletions in FGFR3 have been reported to cause thanatophoric dysplasia types 1 or 2; therefore, this represents the first report to describe such a mutation. © 2016 Wiley Periodicals, Inc.