Characterizing the molecular impact of KMT2D variants on the epigenetic and transcriptional landscapes in Kabuki syndrome.

Kabuki syndrome (KS) is a rare, multisystem disorder with a variable clinical phenotype. The majority of KS is caused by dominant loss-of-function mutations in KMT2D (lysine methyltransferase 2D). KMT2D mediates chromatin accessibility by adding methyl groups to lysine residue 4 of histone 3, which plays a critical role in cell differentiation and homeostasis. The molecular underpinnings of KS remain elusive partly because of a lack of histone modification data from human samples. Consequently, we profiled and characterized alterations in histone modification and gene transcription in peripheral blood mononuclear cells (PBMCs) from 33 patients with KMT2D mutations and 36 unaffected healthy controls. Our analysis identified unique enhancer signatures in H3K4me1 and H3K4me2 in KS compared with controls. Reduced enhancer signals were present for promoter-distal sites of immune-related genes for which co-binding of PBMC-specific transcription factors was predicted; 31% of super-enhancers of normal blood cells overlapped with disrupted enhancers in KS, supporting an association of reduced enhancer activity of immune-related genes with immune deficiency phenotypes. In contrast, increased enhancer signals were observed for promoter-proximal regions of metabolic genes enriched with EGR1 and E2F2 motifs, whose transcriptional levels were significantly increased in KS. Additionally, we identified ~100 de novo enhancers in genes, such as in MYO1F and AGAP2. Together, our results underscore the effect of KMT2D haploinsufficiency on dysregulation of enhancer states and gene transcription and provide a framework for the identification of therapeutic targets and biomarkers in preparation for clinical trial readiness.

Variant Classification for Pompe disease; ACMG/AMP specifications from the ClinGen Lysosomal Diseases Variant Curation Expert Panel.

Accurate determination of the clinical significance of genetic variants is critical to the integration of genomics in medicine. To facilitate this process, the NIH-funded Clinical Genome Resource (ClinGen) has assembled Variant Curation Expert Panels (VCEPs), groups of experts and biocurators which provide gene- and disease- specifications to the American College of Medical Genetics & Genomics and Association for Molecular Pathology's (ACMG/AMP) variation classification guidelines. With the goal of classifying the clinical significance of GAA variants in Pompe disease (Glycogen storage disease, type II), the ClinGen Lysosomal Diseases (LD) VCEP has specified the ACMG/AMP criteria for GAA. Variant classification can play an important role in confirming the diagnosis of Pompe disease as well as in the identification of carriers. Furthermore, since the inclusion of Pompe disease on the Recommended Uniform Screening Panel (RUSP) for newborns in the USA in 2015, the addition of molecular genetic testing has become an important component in the interpretation of newborn screening results, particularly for asymptomatic individuals. To date, the LD VCEP has submitted classifications and supporting data on 243 GAA variants to public databases, specifically ClinVar and the ClinGen Evidence Repository. Here, we describe the ACMG/AMP criteria specification process for GAA, an update of the GAA-specific variant classification guidelines, and comparison of the ClinGen LD VCEP's GAA variant classifications with variant classifications submitted to ClinVar. The LD VCEP has added to the publicly available knowledge on the pathogenicity of variants in GAA by increasing the number of expert-curated GAA variants present in ClinVar, and aids in resolving conflicting classifications and variants of uncertain clinical significance.

ZMIZ1 Variants Cause a Syndromic Neurodevelopmental Disorder.

ZMIZ1 is a coactivator of several transcription factors, including p53, the androgen receptor, and NOTCH1. Here, we report 19 subjects with intellectual disability and developmental delay carrying variants in ZMIZ1. The associated features include growth failure, feeding difficulties, microcephaly, facial dysmorphism, and various other congenital malformations. Of these 19, 14 unrelated subjects carried de novo heterozygous single-nucleotide variants (SNVs) or single-base insertions/deletions, 3 siblings harbored a heterozygous single-base insertion, and 2 subjects had a balanced translocation disrupting ZMIZ1 or involving a regulatory region of ZMIZ1. In total, we identified 13 point mutations that affect key protein regions, including a SUMO acceptor site, a central disordered alanine-rich motif, a proline-rich domain, and a transactivation domain. All identified variants were absent from all available exome and genome databases. In vitro, ZMIZ1 showed impaired coactivation of the androgen receptor. In vivo, overexpression of ZMIZ1 mutant alleles in developing mouse brains using in utero electroporation resulted in abnormal pyramidal neuron morphology, polarization, and positioning, underscoring the importance of ZMIZ1 in neural development and supporting mutations in ZMIZ1 as the cause of a rare neurodevelopmental syndrome.

Lysine methyltransferase 2D regulates muscle fiber size and muscle cell differentiation.

Kabuki syndrome (KS) is a rare genetic disorder caused primarily by mutations in the histone modifier genes KMT2D and KDM6A. The genes have broad temporal and spatial expression in many organs, resulting in complex phenotypes observed in KS patients. Hypotonia is one of the clinical presentations associated with KS, yet detailed examination of skeletal muscle samples from KS patients has not been reported. We studied the consequences of loss of KMT2D function in both mouse and human muscles. In mice, heterozygous loss of Kmt2d resulted in reduced neuromuscular junction (NMJ) perimeter, decreased muscle cell differentiation in vitro and impaired myofiber regeneration in vivo. Muscle samples from KS patients of different ages showed presence of increased fibrotic tissue interspersed between myofiber fascicles, which was not seen in mouse muscles. Importantly, when Kmt2d-deficient muscle stem cells were transplanted in vivo in a physiologic non-Kabuki environment, their differentiation potential is restored to levels undistinguishable from control cells. Thus, the epigenetic changes due to loss of function of KMT2D appear reversible through a change in milieu, opening a potential therapeutic avenue.

De novo loss-of-function KCNMA1 variants are associated with a new multiple malformation syndrome and a broad spectrum of developmental and neurological phenotypes.

KCNMA1 encodes the large-conductance Ca2+- and voltage-activated K+ (BK) potassium channel α-subunit, and pathogenic gain-of-function variants in this gene have been associated with a dominant form of generalized epilepsy and paroxysmal dyskinesia. Here, we genetically and functionally characterize eight novel loss-of-function (LoF) variants of KCNMA1. Genome or exome sequencing and the participation in the international Matchmaker Exchange effort allowed for the identification of novel KCNMA1 variants. Patch clamping was used to assess functionality of mutant BK channels. The KCNMA1 variants p.(Ser351Tyr), p.(Gly356Arg), p.(Gly375Arg), p.(Asn449fs) and p.(Ile663Val) abolished the BK current, whereas p.(Cys413Tyr) and p.(Pro805Leu) reduced the BK current amplitude and shifted the activation curves toward positive potentials. The p.(Asp984Asn) variant reduced the current amplitude without affecting kinetics. A phenotypic analysis of the patients carrying the recurrent p.(Gly375Arg) de novo missense LoF variant revealed a novel syndromic neurodevelopmental disorder associated with severe developmental delay, visceral and cardiac malformations, connective tissue presentations with arterial involvement, bone dysplasia and characteristic dysmorphic features. Patients with other LoF variants presented with neurological and developmental symptoms including developmental delay, intellectual disability, ataxia, axial hypotonia, cerebral atrophy and speech delay/apraxia/dysarthria. Therefore, LoF KCNMA1 variants are associated with a new syndrome characterized by a broad spectrum of neurological phenotypes and developmental disorders. LoF variants of KCNMA1 cause a new syndrome distinctly different from gain-of-function variants in the same gene.

Prenatal and perinatal history in Kabuki Syndrome.

Kabuki syndrome (KS) is a disorder of epigenetic dysregulation due to heterozygous mutations in KMT2D or KDM6A, genes encoding a lysine-specific methyltransferase or demethylase, respectively. The phenotype is highly variable, including congenital cardiac and renal anomalies, developmental delay, hypotonia, failure to thrive, short stature, and immune dysfunction. All affected individuals have characteristic facial features. As KS natural history has not been fully delineated, limited information exists on its prenatal and perinatal history. Two tertiary centers collected retrospective data from individuals with KS (N = 49) using a questionnaire followed by review of medical records. Data from 49 individuals (age range: 7 months-33 years; 37% male; 36 with KMT2D mutations, 2 with KDM6A mutations, and 11 diagnosed clinically) were examined. Polyhydramnios affected 16 of 39 (41%) pregnancies. Abnormal quad screens in four out of nine (44%) pregnancies and reduced placental weights also complicated KS pregnancies. These data comprise the first large dataset on prenatal and perinatal history in individuals with confirmed (genetically or clinically) KS. Over a third of pregnancies were complicated by polyhydramnios, possibly secondary to abnormal craniofacial structures and functional impairment of swallowing. The differential diagnosis for polyhydramnios in the absence of intrauterine growth retardation should include KS.

The phenotypic spectrum of Kabuki syndrome in patients of Chinese descent: A case series.

Kabuki syndrome (KS) is a rare disorder of transcriptional regulation with a complex phenotype that includes cranio-facial dysmorphism, intellectual disability, hypotonia, failure to thrive, short stature, and cardiac and renal anomalies. Heterozygous, de novo dominant mutations in either KMT2D or KDM6A underlie KS. Limited information is available about the phenotypic spectrum of KS in China. Fourteen Chinese patients with genetically confirmed KS were evaluated in addition to 11 Chinese patients who were identified from the medical literature. The clinical phenotype spectrum of these patients was compared to that of 449 patients with KS from non-Chinese ethnicities. Additionally, we explored the utility of a facial recognition software in recognizing KS. All 25 patients with KS carried de novo, likely pathogenic or pathogenic variants in either KMT2D or KDM6A. Eighteen patients were male, the age at diagnosis ranged from 2months to 11.6 years. The facial gestalt included arched and broad eyebrows (25/25; 100%), sparse lateral or notched eyebrows (18/18; 100%), short columella with a concave nasal tip (24/25; 96%) and large, prominent ears (24/24; 100%) which were more frequent in Chinese patients (p < .01). In contrast, microcephaly (2/25; 8%), cleft lip/palate (2/25; 8%), and cardiac defects (10/25; 40%) were less frequent in Chinese patients (not significant). The diagnosis of KS was correctly identified in 13 of 14 patients through facial recognition and clinical phenotyping, underscoring the utility of this approach. As expected, there is marked phenotypic overlap between Chinese and non-Chinese patients with KS, although subtle differences were identified.

A novel FAM20C mutation causes a rare form of neonatal lethal Raine syndrome.

Raine syndrome is a rare, autosomal recessive, osteosclerotic bone dysplasia due to pathogenic variants in FAM20C. The clinical phenotype is characterized by generalized osteosclerosis affecting all bones, cerebral calcifications, and craniofacial dysmorphism. Most cases present during the neonatal period with early lethality due to pulmonary hypoplasia and respiratory compromise while only few affected individuals have been reported to survive into adulthood. FAM20C is a ubiquitously expressed protein kinase that contains five functional domains including a catalytic domain, a binding pocket for FAM20A and three distinct N-glycosylation sites. We report a newborn infant with a history of prenatal onset fractures, generalized osteosclerosis, and craniofacial dysmorphism and early lethality. The clinical presentation was highly suggestive of Raine syndrome. A homozygous, novel missense variant in exon 5 of FAM20C (c.1007T>G; p.Met336Arg) was identified by targeted Sanger sequencing. Following in silico analysis and mapping of the variant on a three-dimensional (3D) model of FAM20C it is predicted to be deleterious and to affect N-glycosylation, protein folding, and subsequent secretion of FAM20C. In addition, we reviewed all published FAM20C mutations and observed that most pathogenic variants affect functional regions within the protein establishing evidence for an emerging genotype-phenotype correlation.

Spectrum of neurodevelopmental disease associated with the GNAO1 guanosine triphosphate-binding region.

OBJECTIVE: To characterize the phenotypic spectrum associated with GNAO1 variants and establish genotype-protein structure-phenotype relationships. METHODS: We evaluated the phenotypes of 14 patients with GNAO1 variants, analyzed their variants for potential pathogenicity, and mapped them, along with those in the literature, on a three-dimensional structural protein model. RESULTS: The 14 patients in our cohort, including one sibling pair, had 13 distinct, heterozygous GNAO1 variants classified as pathogenic or likely pathogenic. We attributed the same variant in two siblings to parental mosaicism. Patients initially presented with seizures beginning in the first 3 months of life (8/14), developmental delay (4/14), hypotonia (1/14), or movement disorder (1/14). All patients had hypotonia and developmental delay ranging from mild to severe. Nine had epilepsy, and nine had movement disorders, including dystonia, ataxia, chorea, and dyskinesia. The 13 GNAO1 variants in our patients are predicted to result in amino acid substitutions or deletions in the GNAO1 guanosine triphosphate (GTP)-binding region, analogous to those in previous publications. Patients with variants affecting amino acids 207-221 had only movement disorder and hypotonia. Patients with variants affecting the C-terminal region had the mildest phenotypes. SIGNIFICANCE: GNAO1 encephalopathy most frequently presents with seizures beginning in the first 3 months of life. Concurrent movement disorders are also a prominent feature in the spectrum of GNAO1 encephalopathy. All variants affected the GTP-binding domain of GNAO1, highlighting the importance of this region for G-protein signaling and neurodevelopment.

Rare variants in SOS2 and LZTR1 are associated with Noonan syndrome.

BACKGROUND: Noonan syndrome is an autosomal dominant, multisystemic disorder caused by dysregulation of the RAS/mitogen activated protein kinase (MAPK) pathway. Heterozygous, pathogenic variants in 11 known genes account for approximately 80% of cases. The identification of novel genes associated with Noonan syndrome has become increasingly challenging, since they might be responsible for very small fractions of the cases. METHODS: A cohort of 50 Brazilian probands negative for pathogenic variants in the known genes associated with Noonan syndrome was tested through whole-exome sequencing along with the relatives in the familial cases. Families from the USA and Poland with mutations in the newly identified genes were included subsequently. RESULTS: We identified rare, segregating or de novo missense variants in SOS2 and LZTR1 in 4% and 8%, respectively, of the 50 Brazilian probands. SOS2 and LZTR1 variants were also found to segregate in one American and one Polish family. Notably, SOS2 variants were identified in patients with marked ectodermal involvement, similar to patients with SOS1 mutations. CONCLUSIONS: We identified two novel genes, SOS2 and LZTR1, associated with Noonan syndrome, thereby expanding the molecular spectrum of RASopathies. Mutations in these genes are responsible for approximately 3% of all patients with Noonan syndrome. While SOS2 is a natural candidate, because of its homology with SOS1, the functional role of LZTR1 in the RAS/MAPK pathway is not known, and it could not have been identified without the large pedigrees. Additional functional studies are needed to elucidate the role of LZTR1 in RAS/MAPK signalling and in the pathogenesis of Noonan syndrome.

Lyso-globotriaosylsphingosine (lyso-Gb3) levels in neonates and adults with the Fabry disease later-onset GLA IVS4+919G>A mutation.

Lyso-globotriaosylsphingosine (lyso-Gb3) is a useful biomarker in the diagnosis and monitoring of treatment for Fabry disease. However, it is unclear whether lyso-Gb3 is elevated in patients with later-onset Fabry disease. Thus, we measured lyso-Gb3 levels from dried blood spots (DBS) from male newborns with the Fabry disease later-onset phenotype, IVS4+919G>A mutation, and their family members. The lyso-Gb3 levels were below the detection limit in normal control newborns and were slightly higher in adults. In males of all ages with the IVS4+919G>A mutation, lyso-Gb3 levels were elevated and were higher than in age-matched controls. The elevation of lyso-Gb3 levels in males with the IVS4+919G>A mutation was only slightly elevated compared with patients with the classical Fabry phenotype. The measurement of lyso-Gb3 levels is useful in the diagnosis of Fabry disease, including the later-onset phenotype. The DBS lyso-Gb3 level was not elevated in IVS4+919G>A heterozygotes, and is not useful for their diagnosis. Since lyso-Gb3 levels are elevated from birth in Fabry disease males, "an elevated lyso-Gb3 level" may be of little values for deciding when to begin enzyme replacement therapy.

From Genotype to Phenotype-A Review of Kabuki Syndrome.

Kabuki syndrome (KS) is a rare neuro-developmental disorder caused by variants in genes of histone modification, including KMT2D and KDM6A. This review assesses our current understanding of KS, which was originally named Niikawa-Kuroki syndrome, and aims to guide surveillance and medical care of affected individuals as well as identify gaps in knowledge and unmet patient needs. Ovid MEDLINE and EMBASE databases were searched from 1981 to 2021 to identify reports related to genotype and systems-based phenotype characterization of KS. A total of 2418 articles were retrieved, and 152 were included in this review, representing a total of 1369 individuals with KS. Genotype, phenotype, and the developmental and behavioral profile of KS are reviewed. There is a continuous clinical phenotype spectrum associated with KS with notable variability between affected individuals and an emerging genotype-phenotype correlation. The observed clinical variability may be attributable to differences in genotypes and/or unknown genetic and epigenetic factors. Clinical management is symptom oriented, fragmented, and lacks established clinical care standards. Additional research should focus on enhancing understanding of the burden of illness, the impact on quality of life, the adult phenotype, life expectancy and development of standard-of-care guidelines.

Laboratory and genetic evaluation of Gaucher disease.

Gaucher disease (GD) is an inherited lysosomal storage disorder due to deficiency of glucocerebrosidase. Diagnosis of GD may be suspected based on clinical symptoms and confirmed by the analysis of glucocerebrosidase in total white cells, mononuclear cells, fibroblasts and dried blood on filter paper. Low enzyme activities should be followed by molecular analysis of the GBA gene. Although there is no obvious genotype-phenotype correlation, the presence of p.N370S protects from neurological involvement whereas homozygosity of p.L444P mostly leads to a neuronopathic form of GD. Progressive storage of glucosylceramide in mononuclear cells and macrophages results in elevated levels of chitotriosidase and CCL18/PARC which may be used as biomarker to assess disease severity and efficacy of treatment. Chitotriosidase activities cannot be analysed in at least 6% of GD patients due to a null mutation in the corresponding gene.

The tale of two genes: from next-generation sequencing to phenotype.

An 18-yr-old man with a history of intellectual disability, craniofacial dysmorphism, seizure disorder, and obesity was identified to carry a de novo, pathogenic variant in ASXL1 (c.4198G>T; p.E1400X) associated with the diagnosis of Bohring-Opitz syndrome based on exome sequencing. In addition, he was identified to carry a maternally inherited and likely pathogenic variant in MC4R (c.817C>T; p.Q273X) associated with monogenic obesity. Dual genetic diagnosis occurs in 4%-6% of patients and results in unique clinical phenotypes that are a function of tissue-specific gene expression, involved pathways, clinical expressivity, and penetrance. This case highlights the utility of next-generation sequencing in patients with an unusual combination of clinical presentations for several pillars of precision medicine including (1) diagnosis, (2) prognosis and outcome, (3) management and therapy, and (4) utilization of resources.

Transcobalamin II deficiency at birth.

Transcobalamin II deficiency (# MIM 275350) is a rare, recessively inherited disorder of cobalamin transport that leads to intracellular cobalamin depletion with secondary impairment of methionine synthetase and methyl-malonyl CoA mutase activities. Affected individuals may suffer from long-term neurological sequelae if therapy with intramuscular hydroxocobalamin is not initiated promptly. We report two sisters with complete absence of transcobalamin due to homozygosity for a novel mutation (c.insC110) in the TCN2 gene that leads to a premature stop codon and non-functional protein. The older sister, now 4.5 years old, presented at 6 weeks of age with pancytopenia, protein losing enteropathy and a rapidly declining clinical course. Prompt therapy with 1mg hydroxocobalamin/day led to full recovery within days. Her now 1.5 year old sister was diagnosed shortly after birth and was started on hydroxocobalamin prior to onset of clinical symptoms. Interestingly, urinary methylmalonic acid excretion was increased significantly during the first days of life suggesting that functional cobalamin deficiency is present also during fetal life, although not giving rise to clinical symptoms until well after birth.

Birth weight in patients with mucopolysaccharidosis type II: Data from the Hunter Outcome Survey (HOS).

There is a need to identify early disease markers to facilitate diagnosis of mucopolysaccharidosis type II (MPS II; Hunter syndrome). Mean birth weight and its association with disease severity was investigated in 609 patients enrolled in the Hunter Outcome Survey (HOS). This analysis indicated that birth weight is not an early marker of MPS II and is not associated with disease severity. It remains important to investigate the utility of other factors for early/pre-symptomatic diagnosis.

Utility of rapid whole-exome sequencing in the diagnosis of Niemann-Pick disease type C presenting with fetal hydrops and acute liver failure.

Rapid whole-exome sequencing (rWES) is used in critically ill newborn infants to inform about diagnosis, clinical management, and prognosis. Here we report a male newborn infant with hydrops, pancytopenia, and acute liver failure who was listed for liver transplantation. Given the acuity of the presentation, the procedure-related morbidity and mortality, and lack of diagnosis, we used rWES in the proband and both parents with a turnaround time of 10 business days. rWES returned one maternally inherited, likely pathogenic and one paternally inherited, likely pathogenic variant in NPC1, suggestive of a diagnosis of Niemann-Pick disease type C (NPC). Interestingly, a diagnosis of NPC was entertained prior to rWES, but deemed unlikely in light of absent cholesterol storage on liver biopsy and near-normal oxysterol levels in dried blood. The diagnosis of NPC was confirmed on filipin stain in fibroblasts demonstrating defective cholesterol trafficking. NPC is a slowly progressive neurodegenerative disorder that may also affect the liver with overall poor prognosis. It was decided to take the infant off the transplant list and transfer to palliative care, where he died after 4 wk. This case highlights the utility of rWES in an acute clinical setting for several domains of precision medicine including (1) diagnosis, (2) prognosis and outcome, (3) management and therapy, and (4) utilization of resources.

A defect in the inner kinetochore protein CENPT causes a new syndrome of severe growth failure.

Primordial growth failure has been linked to defects in the biology of cell division and replication. The complex processes involved in microtubule spindle formation, organization and function have emerged as a dominant patho-mechanism in these conditions. The majority of reported disease genes encode for centrosome and centriole proteins, leaving kinetochore proteins by which the spindle apparatus interacts with the chromosomes largely unaccounted for. We report a novel disease gene encoding the constitutive inner kinetochore member CENPT, which is involved in kinetochore targeting and assembly, resulting in severe growth failure in two siblings of a consanguineous family. We herein present studies on the molecular and cellular mechanisms that explain how genetic mutations in this gene lead to primordial growth failure. In both, affected human cell lines and a zebrafish knock-down model of Cenpt, we observed aberrations in cell division with abnormal accumulation of micronuclei and of nuclei with increased DNA content arising from incomplete and/or irregular chromosomal segregation. Our studies underscore the critical importance of kinetochore function for overall body growth and provide new insight into the cellular mechanisms implicated in the spectrum of these severe growth disorders.

Mutations in SLC39A14 disrupt manganese homeostasis and cause childhood-onset parkinsonism-dystonia.

Although manganese is an essential trace metal, little is known about its transport and homeostatic regulation. Here we have identified a cohort of patients with a novel autosomal recessive manganese transporter defect caused by mutations in SLC39A14. Excessive accumulation of manganese in these patients results in rapidly progressive childhood-onset parkinsonism-dystonia with distinctive brain magnetic resonance imaging appearances and neurodegenerative features on post-mortem examination. We show that mutations in SLC39A14 impair manganese transport in vitro and lead to manganese dyshomeostasis and altered locomotor activity in zebrafish with CRISPR-induced slc39a14 null mutations. Chelation with disodium calcium edetate lowers blood manganese levels in patients and can lead to striking clinical improvement. Our results demonstrate that SLC39A14 functions as a pivotal manganese transporter in vertebrates.