Novel insights into the phenotypic spectrum and pathogenesis of Hardikar syndrome.

PURPOSE: Hardikar syndrome (HS, MIM #301068) is a female-specific multiple congenital anomaly syndrome characterized by retinopathy, orofacial clefting, aortic coarctation, biliary dysgenesis, genitourinary malformations, and intestinal malrotation. We previously showed that heterozygous nonsense and frameshift variants in MED12 cause HS. The phenotypic spectrum of disease and the mechanism by which MED12 variants cause disease is unknown. We aim to expand the phenotypic and molecular landscape of HS and elucidate the mechanism by which MED12 variants cause disease. METHODS: We assembled and clinically and molecularly characterized a cohort of 11 previously-unreported individuals with HS. We additionally studied the effect of MED12 deficiency on ciliary biology and hedgehog and YAP signaling, pathways implicated in diseases with phenotypic overlap with HS. RESULTS: We report novel phenotypes associated with HS, including cardiomyopathy, arrhythmia, and vascular anomalies and expand the molecular landscape of HS to include splice site variants. We additionally demonstrate that MED12 deficiency causes decreased cell ciliation and impairs hedgehog and YAP signaling. CONCLUSION: Our data support updating HS standard-of-care to include regular cardiac imaging, arrhythmia screening, and vascular imaging. We further propose that dysregulation of ciliogenesis and YAP and hedgehog signaling contributes to the pathogenesis of HS.

Secondary ACMG and non-ACMG genetic findings in a multiethnic cohort of 16,713 pediatric participants.

PURPOSE: Clinical next-generation sequencing is an effective approach for identifying pathogenic sequence variants that are medically actionable for participants and families but are not associated with the participant's primary diagnosis. These variants are called secondary findings (SFs). According to the literature, there is no report of the types and frequencies of SFs in a large pediatric cohort which includes substantial African-American participants. We sought to investigate the types (including American College of Medical Genetics and Genomics [ACMG] and non-ACMG recommended gene lists), frequencies, and rates of SFs, as well as the effects of SF disclosure on the participants and families of a large pediatric cohort at the Center for Applied Genomics at The Children's Hospital of Philadelphia (CHOP). METHODS: We systematically identified pathogenic (P) and likely pathogenic (LP) variants in established disease-causing genes, adhering to ACMG v3.2 secondary finding guidelines and beyond. For non-ACMG secondary findings, akin to incidental findings in clinical settings, we utilized a set of criteria focusing on pediatric onset, high penetrance, moderate to severe phenotypes, and the clinical actionability of the variants. This criteria-based approach was applied rather than using a fixed gene list to ensure that the variants identified are likely to impact participant health significantly. To identify and categorize these variants, we employed a clinical-grade variant classification standard per ACMG/AMP recommendations; additionally, we conducted a detailed literature search to ensure a comprehensive exploration of potential secondary findings relevant to pediatric participants. RESULTS: We report a distinctive distribution of 1,464 P/LP SF variants in 16,713 participants. There were 427 unique variants in ACMG genes and 265 in non-ACMG genes. The most frequently mutated genes among the ACMG and non-ACMG gene lists were TTR (41.6%) and CHEK2 (7.16%), respectively. Overall, variants of possible medical importance were found in 8.76% of participants in both ACMG (5.81%) and non-ACMG (2.95%) genes.

De novo variants in RNF213 are associated with a clinical spectrum ranging from Leigh syndrome to early-onset stroke.

PURPOSE: RNF213, encoding a giant E3 ubiquitin ligase, has been recognized for its role as a key susceptibility gene for moyamoya disease. Case reports have also implicated specific variants in RNF213 with an early-onset form of moyamoya disease with full penetrance. We aimed to expand the phenotypic spectrum of monogenic RNF213-related disease and to evaluate genotype-phenotype correlations. METHODS: Patients were identified through reanalysis of exome sequencing data of an unselected cohort of unsolved pediatric cases and through GeneMatcher or ClinVar. Functional characterization was done by proteomics analysis and oxidative phosphorylation enzyme activities using patient-derived fibroblasts. RESULTS: We identified 14 individuals from 13 unrelated families with (de novo) missense variants in RNF213 clustering within or around the Really Interesting New Gene (RING) domain. Individuals presented either with early-onset stroke (n = 11) or with Leigh syndrome (n = 3). No genotype-phenotype correlation could be established. Proteomics using patient-derived fibroblasts revealed no significant differences between clinical subgroups. 3D modeling revealed a clustering of missense variants in the tertiary structure of RNF213 potentially affecting zinc-binding suggesting a gain-of-function or dominant negative effect. CONCLUSION: De novo missense variants in RNF213 clustering in the E3 RING or other regions affecting zinc-binding lead to an early-onset syndrome characterized by stroke or Leigh syndrome.

Patent ductus arteriosus and coarctation of the aorta in association with PRDM6 variants.

Patent ductus arteriosus (PDA) and coarctation of the aorta (CoA) are relatively common congenital heart defects. Pathogenic variants in PRDM6, which encodes a smooth-muscle-cell-specific transcription factor, have now been etiologically associated with non-syndromic PDA. We present three patients with PDA and CoA found to harbor PRDM6 variants, including a novel, likely-pathogenic variant.

Combined PMM2-CDG and hereditary fructose intolerance in a patient with mild clinical presentation.

We report a patient with an extremely rare, combined diagnosis of PMM2-CDG and hereditary fructose intolerance (HFI). By comparing with other patients, under-galactosylation was identified as a feature of HFI. Fructose/sorbitol/sucrose restriction was initiated right afterwards. The patient is at the mild end of the PMM2-CDG spectrum, raising the question of sorbitol's role in the pathogenesis of PMM2-CDG and whether fructose/sorbitol/sucrose restriction could benefit other PMM2-CDG patients. Additionally, epalrestat, an emerging potential PMM2-CDG therapy, may benefit HFI patients.

TOPORS as a novel causal gene for Joubert syndrome.

Joubert syndrome (JBTS) is a Mendelian disorder of the primary cilium defined by the clinical triad of hypotonia, developmental delay, and a distinct cerebellar malformation called the molar tooth sign. JBTS is inherited in an autosomal recessive, autosomal dominant, or X-linked recessive manner. Though over 40 genes have been identified as causal for JBTS, molecular diagnosis is not made in 30%-40% of individuals who meet clinical criteria. TOPORS encodes topoisomerase I-binding arginine/serine-rich protein, and homozygosity for a TOPORS missense variant (c.29C > A; p.(Pro10Gln)) was identified in individuals with the ciliopathy oral-facial-digital syndrome in two families of Dominican descent. Here, we report an additional proband of Dominican ancestry with JBTS found by exome sequencing to be homozygous for the identical p.(Pro10Gln) TOPORS missense variant. Query of the Mount Sinai BioMe biobank, which includes 1880 individuals of Dominican ancestry, supports a high carrier frequency of the TOPORS p.(Pro10Gln) variant in individuals of Dominican descent. Our data nominates TOPORS as a novel causal gene for JBTS and suggests that TOPORS variants should be considered in the differential of ciliopathy-spectrum disease in individuals of Dominican ancestry.

A mutational hotspot in AMOTL1 defines a new syndrome of orofacial clefting, cardiac anomalies, and tall stature.

AMOTL1 encodes angiomotin-like protein 1, an actin-binding protein that regulates cell polarity, adhesion, and migration. The role of AMOTL1 in human disease is equivocal. We report a large cohort of individuals harboring heterozygous AMOTL1 variants and define a core phenotype of orofacial clefting, congenital heart disease, tall stature, auricular anomalies, and gastrointestinal manifestations in individuals with variants in AMOTL1 affecting amino acids 157-161, a functionally undefined but highly conserved region. Three individuals with AMOTL1 variants outside this region are also described who had variable presentations with orofacial clefting and multi-organ disease. Our case cohort suggests that heterozygous missense variants in AMOTL1, most commonly affecting amino acid residues 157-161, define a new orofacial clefting syndrome, and indicates an important functional role for this undefined region.

The current state of adult metabolic medicine in the United States: Results of a nationwide survey.

PURPOSE: Patients with inherited metabolic disorders (IMDs) now have improved health outcomes and increased survival into adulthood. There is scant evidence on managing adults with IMDs. We present an analysis of current care practices for adults with IMDs in the United States. METHODS: We created and distributed an online survey to US members of the Society of Inherited Metabolic Disorders. The survey addressed ambulatory care, acute management, and health care transition (HCT) practices of adults with IMDs. RESULTS: The survey was completed by 91 providers from 73 institutions. Most adult patients with IMDs receive lifelong care from a single metabolic clinician, predominantly in pediatric clinic settings. Adults receive comprehensive ambulatory metabolic care, but fewer trainees participate compared with pediatric visits. Most acute IMD management occurs in pediatric hospitals. Clinician comfort with HCT increased the frequency of HCT planning. Overall, all respondents felt that providing specialized care to adults with IMDs is high value. CONCLUSION: Our survey demonstrates the paucity of clinical resources dedicated to adult metabolic medicine. Care is fragmented and varies by medical system. Interest in HCT is robust but would benefit from standardized practices. Our findings reinforce the need for greater focus on adult metabolic medicine in the United States.

Stankiewicz-Isidor syndrome: expanding the clinical and molecular phenotype.

PURPOSE: Haploinsufficiency of PSMD12 has been reported in individuals with neurodevelopmental phenotypes, including developmental delay/intellectual disability (DD/ID), facial dysmorphism, and congenital malformations, defined as Stankiewicz-Isidor syndrome (STISS). Investigations showed that pathogenic variants in PSMD12 perturb intracellular protein homeostasis. Our objective was to further explore the clinical and molecular phenotypic spectrum of STISS. METHODS: We report 24 additional unrelated patients with STISS with various truncating single nucleotide variants or copy-number variant deletions involving PSMD12. We explore disease etiology by assessing patient cells and CRISPR/Cas9-engineered cell clones for various cellular pathways and inflammatory status. RESULTS: The expressivity of most clinical features in STISS is highly variable. In addition to previously reported DD/ID, speech delay, cardiac and renal anomalies, we also confirmed preaxial hand abnormalities as a feature of this syndrome. Of note, 2 patients also showed chilblains resembling signs observed in interferonopathy. Remarkably, our data show that STISS patient cells exhibit a profound remodeling of the mTORC1 and mitophagy pathways with an induction of type I interferon-stimulated genes. CONCLUSION: We refine the phenotype of STISS and show that it can be clinically recognizable and biochemically diagnosed by a type I interferon gene signature.

Delayed diagnosis and racial bias in children with genetic conditions.

As more therapeutics for genetic conditions become available, the need for timely and equitable genetic diagnosis has become urgent. Using clinical cases, we consider the health system-, provider-, and patient-level factors that contribute to the delayed diagnosis of genetic conditions in pediatric patients from minority populations, leading to health disparities between racial groups. We then provide suggestions to address these factors, with the aim of improving minority health and access to genetic care for all children.

TBX6 as a cause of a combined skeletal-kidney dysplasia syndrome.

TBX6 encodes transcription-factor box 6, a transcription factor critical to paraxial mesoderm segmentation and somitogenesis during embryonic development. TBX6 haploinsufficiency is believed to drive the skeletal and kidney phenotypes associated with the 16p11.2 deletion syndrome. Heterozygous and biallelic variants in TBX6 are associated with vertebral and rib malformations (TBX6-associated congenital scoliosis) and spondylocostal dysostosis, and heterozygous TBX6 variants are associated with increased risk of genitourinary tract malformations. Combined skeletal and kidney phenotypes in individuals harboring heterozygous or biallelic TBX6 variants are rare. Here, we present seven individuals with vertebral and rib malformations and structural kidney differences associated with heterozygous TBX6 gene deletion in trans with a hypomorphic TBX6 allele or biallelic TBX6 variants. Our case series highlights the association between TBX6 and both skeletal and kidney disease.

A novel MBTPS2 variant associated with BRESHECK syndrome impairs sterol-regulated transcription and the endoplasmic reticulum stress response.

Ichthyosis follicularis, atrichia, and photophobia syndrome (IFAP syndrome) is a rare, X-linked disorder caused by pathogenic variants in membrane-bound transcription factor protease, site 2 (MBTPS2). Pathogenic MBTPS2 variants also cause BRESHECK syndrome, characterized by the IFAP triad plus intellectual disability and multiple congenital anomalies. Here we present a patient with ichthyosis, sparse hair, pulmonic stenosis, kidney dysplasia, hypospadias, growth failure, thrombocytopenia, anemia, bone marrow fibrosis, and chronic diarrhea found by research-based exome sequencing to harbor a novel, maternally inherited MBTPS2 missense variant (c.766 G>A; (p.Val256Leu)). In vitro modeling supports variant pathogenicity, with impaired cell growth in cholesterol-depleted media, attenuated activation of the sterol regulatory element-binding protein pathway, and failure to activate the endoplasmic reticulum stress response pathway. Our case expands both the genetic and phenotypic spectrum of BRESHECK syndrome to include a novel MBTPS2 variant and cytopenias, bone marrow fibrosis, and chronic diarrhea.

De novo loss-of-function variants in X-linked MED12 are associated with Hardikar syndrome in females.

PURPOSE: Hardikar syndrome (MIM 612726) is a rare multiple congenital anomaly syndrome characterized by facial clefting, pigmentary retinopathy, biliary anomalies, and intestinal malrotation, but with preserved cognition. Only four patients have been reported previously, and none had a molecular diagnosis. Our objective was to identify the genetic basis of Hardikar syndrome (HS) and expand the phenotypic spectrum of this disorder. METHODS: We performed exome sequencing on two previously reported and five unpublished female patients with a clinical diagnosis of HS. X-chromosome inactivation (XCI) studies were also performed. RESULTS: We report clinical features of HS with previously undescribed phenotypes, including a fatal unprovoked intracranial hemorrhage at age 21. We additionally report the discovery of de novo pathogenic nonsense and frameshift variants in MED12 in these seven individuals and evidence of extremely skewed XCI in all patients with informative testing. CONCLUSION: Pathogenic missense variants in the X-chromosome gene MED12 have previously been associated with Opitz-Kaveggia syndrome, Lujan syndrome, Ohdo syndrome, and nonsyndromic intellectual disability, primarily in males. We propose a fifth, female-specific phenotype for MED12, and suggest that nonsense and frameshift loss-of-function MED12 variants in females cause HS. This expands the MED12-associated phenotype in females beyond intellectual disability.

Rare variants in KDR, encoding VEGF Receptor 2, are associated with tetralogy of Fallot.

PURPOSE: Rare genetic variants in KDR, encoding the vascular endothelial growth factor receptor 2 (VEGFR2), have been reported in patients with tetralogy of Fallot (TOF). However, their role in disease causality and pathogenesis remains unclear. METHODS: We conducted exome sequencing in a familial case of TOF and large-scale genetic studies, including burden testing, in >1,500 patients with TOF. We studied gene-targeted mice and conducted cell-based assays to explore the role of KDR genetic variation in the etiology of TOF. RESULTS: Exome sequencing in a family with two siblings affected by TOF revealed biallelic missense variants in KDR. Studies in knock-in mice and in HEK 293T cells identified embryonic lethality for one variant when occurring in the homozygous state, and a significantly reduced VEGFR2 phosphorylation for both variants. Rare variant burden analysis conducted in a set of 1,569 patients of European descent with TOF identified a 46-fold enrichment of protein-truncating variants (PTVs) in TOF cases compared to controls (P = 7 × 10-11). CONCLUSION: Rare KDR variants, in particular PTVs, strongly associate with TOF, likely in the setting of different inheritance patterns. Supported by genetic and in vivo and in vitro functional analysis, we propose loss-of-function of VEGFR2 as one of the mechanisms involved in the pathogenesis of TOF.

A novel heterotaxy gene: Expansion of the phenotype of TTC21B-spectrum disease.

TTC21B encodes the protein IFT139, a critical component of the retrograde transport system within the primary cilium. Biallelic, pathogenic TTC21B variants are associated with classic ciliopathy syndromes, including nephronophthisis, Jeune asphyxiating thoracic dystrophy, and Joubert Syndrome, with ciliopathy-spectrum traits such as biliary dysgenesis, primary ciliary dyskinesia, and situs inversus, and also with focal segmental glomerulosclerosis. We report a 9-year-old male with focal segmental glomerulosclerosis requiring kidney transplant, primary ciliary dyskinesia, and biliary dysgenesis, found by research-based exome sequencing to have biallelic pathogenic TTC21B variants. A sibling with isolated heterotaxy was found to harbor the same variants. This case highlights the phenotypic spectrum and unpredictable manifestations of TTC21B-related disease, and also reports the first association between TTC21B and heterotaxy, nominating TTC21B as an important new heterotaxy gene.

Expanding the phenotypic spectrum of Mendelian connective tissue disorders to include prominent kidney phenotypes.

Heritable connective tissue disorders are a group of diseases, each rare, characterized by various combinations of skin, joint, musculoskeletal, organ, and vascular involvement. Although kidney abnormalities have been reported in some connective tissue disorders, they are rarely a presenting feature. Here we present three patients with prominent kidney phenotypes who were found by whole exome sequencing to have variants in established connective tissue genes associated with Loeys-Dietz syndrome and congenital contractural arachnodactyly. These cases highlight the importance of considering connective tissue disease in children presenting with structural kidney disease and also serves to expand the phenotype of Loeys-Dietz syndrome and possibly congenital contractural arachnodactyly to include cystic kidney disease and cystic kidney dysplasia, respectively.

Expanding the genetic landscape of oral-facial-digital syndrome with two novel genes.

Oral-facial-digital syndromes (OFDS) are a heterogeneous and rare group of Mendelian disorders characterized by developmental abnormalities of the oral cavity, face, and digits caused by dysfunction of the primary cilium, a mechanosensory organelle that exists atop most cell types that facilitates organ patterning and growth. OFDS is inherited both in an X-linked dominant, X-linked recessive, and autosomal recessive manner. Importantly, though many of the causal genes for OFDS have been identified, up to 40% of OFD syndromes are of unknown genetic basis. Here we describe three children with classical presentations of OFDS including lingual hamartomas, polydactyly, and characteristic facial features found by exome sequencing to harbor variants in causal genes not previously associated with OFDS. We describe a female with hypothalamic hamartoma, urogenital sinus, polysyndactyly, and multiple lingual hamartomas consistent with OFDVI with biallelic pathogenic variants in CEP164, a gene associated with ciliopathy-spectrum disease, but never before with OFDS. We additionally describe two unrelated probands with postaxial polydactyly, multiple lingual hamartomas, and dysmorphic features both found to be homozygous for an identical TOPORS missense variant, c.29 C>A; (p.Pro10Gln). Heterozygous TOPORS pathogenic gene variants are associated with autosomal dominant retinitis pigmentosa, but never before with syndromic ciliopathy. Of note, both probands are of Dominican ancestry, suggesting a possible founder allele.

Ciliopathies: Coloring outside of the lines.

Ciliopathy syndromes are a diverse spectrum of disease characterized by a combination of cystic kidney disease, hepatobiliary disease, retinopathy, skeletal dysplasia, developmental delay, and brain malformations. Though generally divided into distinct disease categories based on the pattern of system involvement, ciliopathy syndromes are known to display certain phenotypic overlap. We performed next-generation sequencing panel testing, clinical exome sequencing, and research-based exome sequencing reanalysis on patients with suspected ciliopathy syndromes with additional features. We identified biallelic pathogenic variants in BBS1 in a child with features of cranioectodermal dysplasia, and biallelic variants in BBS12 in a child with the clinical stigmata of Bardet-Biedl syndrome, but also with anal atresia. We additionally identified biallelic pathogenic variants in WDR35 and DYNC2H1 in children with predominant liver disease and ductal plate malformation without skeletal dysplasia. Our study highlights the phenotypic and genetic diversity of ciliopathy syndromes, the importance of considering ciliopathy syndromes as a disease-spectrum and screening for all associated complications in all patients, and describes exclusive extra-skeletal manifestations in two classical skeletal dysplasia syndromes.

A new syndrome of moyamoya disease, kidney dysplasia, aminotransferase elevation, and skin disease associated with de novo variants in RNF213.

Ring-finger protein 213 (RNF213) encodes a protein of unknown function believed to play a role in cellular metabolism and angiogenesis. Gene variants are associated with susceptibility to moyamoya disease. Here, we describe two children with moyamoya disease who also demonstrated kidney disease, elevated aminotransferases, and recurrent skin lesions found by exome sequencing to have de novo missense variants in RNF213. These cases highlight the ability of RNF213 to cause Mendelian moyamoya disease in addition to acting as a genetic susceptibility locus. The cases also suggest a new, multi-organ RNF213-spectrum disease characterized by liver, skin, and kidney pathology in addition to severe moyamoya disease caused by heterozygous, de novo C-terminal RNF213 missense variants.

"Blepharophimosis-plus" syndromes: Frequency of systemic genetic disorders that also include blepharophimosis.

BACKGROUND: To determine the frequency of isolated blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) versus systemic genetic disorders in patients presenting with blepharophimosis. METHODS: Retrospective clinical records review. The records of all patients with blepharophimosis seen in the Division of Ophthalmology at the Children's Hospital of Philadelphia during a 12-year-period (2009-2020) were reviewed for medical history, clinical examination findings and results of genetic analyses. RESULTS: The 135 patients identified with blepharophimosis included 72 females (53%) and 63 males (47%) whose mean ± standard deviation age at first visit was 3.5 ± 6.4 years (range 0-39.8 years). Sixty-seven of the patients (50%) had undergone genetic testing for FOXL2 gene mutation. Fifty-four (81%) harboured FOXL2 gene mutations and 13 (19%) did not. Altogether, 126 patients (93%) had a final diagnosis of isolated BPES. The remaining nine (7%) had syndromic diagnoses ("blepharophimosis-plus"), including Dubowitz syndrome (n = 2), Ohdo syndrome (n = 1), 22q11.2 duplication (n = 1) and 3q22 deletion (n = 2). Three patients with multiple congenital anomalies remain undiagnosed. CONCLUSIONS: Blepharophimosis is an eyelid feature occurring most commonly in isolation due to FOXL2 gene mutation, but can also be a harbinger of multisystem disease not exclusive to isolated BPES, as observed in 7% of cases in this series. The ophthalmologist is often the first to recognise these unique features, and must consider and rule out non-BPES syndromes before establishing a diagnosed classic BPES. A comprehensive genetic evaluation is, therefore, indicated in all cases.