Tuberous sclerosis complex: a complex case.

Tuberous sclerosis complex (TSC) is an inheritable disorder characterized by the formation of benign yet disorganized tumors in multiple organ systems. Germline mutations in the TSC1 (hamartin) or more frequently TSC2 (tuberin) genes are causative for TSC. The malignant manifestations of TSC, pulmonary lymphangioleiomyomatosis (LAM) and renal angiomyolipoma (AML), may also occur as independent sporadic perivascular epithelial cell tumor (PEComa) characterized by somatic TSC2 mutations. Thus, discerning TSC from the copresentation of sporadic LAM and sporadic AML may be obscured in TSC patients lacking additional features. In this report, we present a case study on a single patient initially reported to have sporadic LAM and a mucinous duodenal adenocarcinoma deficient in DNA mismatch repair proteins. Moreover, the patient had a history of Wilms' tumor, which was reclassified as AML following the LAM diagnosis. Therefore, we investigated the origins and relatedness of these tumors. Using germline whole-genome sequencing, we identified a premature truncation in one of the patient's TSC2 alleles. Using immunohistochemistry, loss of tuberin expression was observed in AML and LAM tissue. However, no evidence of a somatic loss of heterozygosity or DNA methylation epimutations was observed at the TSC2 locus, suggesting alternate mechanisms may contribute to loss of the tumor suppressor protein. In the mucinous duodenal adenocarcinoma, no causative mutations were found in the DNA mismatch repair genes MLH1, MSH2, MSH6, or PMS2 Rather, clonal deconvolution analyses were used to identify mutations contributing to pathogenesis. This report highlights both the utility of using multiple sequencing techniques and the complexity of interpreting the data in a clinical context.

Intragenic Deletions in FLNB Are Part of the Mutational Spectrum Causing Spondylocarpotarsal Synostosis Syndrome.

Spondylocarpotarsal synostosis syndrome (SCT) is characterized by vertebral fusions, a disproportionately short stature, and synostosis of carpal and tarsal bones. Pathogenic variants in FLNB, MYH3, and possibly in RFLNA, have been reported to be responsible for this condition. Here, we present two unrelated individuals presenting with features typical of SCT in which Sanger sequencing combined with whole genome sequencing identified novel, homozygous intragenic deletions in FLNB (c.1346-1372_1941+389del and c.3127-353_4223-1836del). Both deletions remove several consecutive exons and are predicted to result in a frameshift. To our knowledge, this is the first time that large structural variants in FLNB have been reported in SCT, and thus our findings add to the classes of variation that can lead to this disorder. These cases highlight the need for copy number sensitive methods to be utilized in order to be comprehensive in the search for a molecular diagnosis in individuals with a clinical diagnosis of SCT.

Trans-ancestral dissection of urate- and gout-associated major loci SLC2A9 and ABCG2 reveals primate-specific regulatory effects.

Gout is a complex inflammatory arthritis affecting ~20% of people with an elevated serum urate level (hyperuricemia). Gout and hyperuricemia are essentially specific to humans and other higher primates, with varied prevalence across ancestral groups. SLC2A9 and ABCG2 are major loci associated with both urate and gout in multiple ancestral groups. However, fine mapping has been challenging due to extensive linkage disequilibrium underlying the associated regions. We used trans-ancestral fine mapping integrated with primate-specific genomic information to address this challenge. Trans-ancestral meta-analyses of GWAS cohorts of either European (EUR) or East Asian (EAS) ancestry resulted in single-variant resolution mappings for SLC2A9 (rs3775948 for urate and rs4697701 for gout) and ABCG2 (rs2622621 for gout). Tests of colocalization of variants in both urate and gout suggested existence of a shared candidate causal variant for SLC2A9 only in EUR and for ABCG2 only in EAS. The fine-mapped gout variant rs4697701 was within an ancient enhancer, whereas rs2622621 was within a primate-specific transposable element, both supported by functional evidence from the Roadmap Epigenomics project in human primary tissues relevant to urate and gout. Additional primate-specific elements were found near both loci and those adjacent to SLC2A9 overlapped with known statistical epistatic interactions associated with urate as well as multiple super-enhancers identified in urate-relevant tissues. We conclude that by leveraging ancestral differences trans-ancestral fine mapping has identified ancestral and functional variants for SLC2A9 or ABCG2 with primate-specific regulatory effects on urate and gout.

Wilms tumor in patients with osteopathia striata with cranial sclerosis.

Germline pathogenic variants in AMER1 cause osteopathia striata with cranial sclerosis (OSCS: OMIM 300373), an X-linked sclerosing bone disorder. Female heterozygotes exhibit metaphyseal striations in long bones, macrocephaly, cleft palate, and, occasionally, learning disability. Male hemizygotes typically manifest the condition as fetal or neonatal death. Somatically acquired variants in AMER1 are found in neoplastic tissue in 15-30% of patients with Wilms tumor; however, to date, only one individual with OSCS has been reported with a Wilms tumor. Here we present four cases of Wilms tumor in unrelated individuals with OSCS, including the single previously published case. We also report the first case of bilateral Wilms tumor in a patient with OSCS. Tumor tissue analysis showed no clear pattern of histological subtypes. In Beckwith-Wiedemann syndrome, which has a known predisposition to Wilms tumor development, clinical protocols have been developed for tumor surveillance. In the absence of further evidence, we propose a similar protocol for patients with OSCS to be instituted as an initial precautionary approach to tumor surveillance. Further evidence is needed to refine this protocol and to evaluate the possibility of development of other neoplasms later in life, in patients with OSCS.

Deletion of Exon 1 in AMER1 in Osteopathia Striata with Cranial Sclerosis.

Osteopathia striata with cranial sclerosis (OSCS) is an X-linked dominant condition characterised by metaphyseal striations, macrocephaly, cleft palate, and developmental delay in affected females. Males have a more severe phenotype with multi-organ malformations, and rarely survive. To date, only frameshift and nonsense variants in exon 2, the single coding exon of AMER1, or whole gene deletions have been reported to cause OSCS. In this study, we describe two families with phenotypic features typical of OSCS. Exome sequencing and multiplex ligation-dependent probe amplification (MLPA) did not identify pathogenic variants in AMER1. Therefore, genome sequencing was employed which identified two deletions containing the non-coding exon 1 of AMER1 in the families. These families highlight the importance of considering variants or deletions of upstream non-coding exons in conditions such as OSCS, noting that often such exons are not captured on probe or enrichment-based platforms because of their high G/C content.

An Activating Variant in CTNNB1 is Associated with a Sclerosing Bone Dysplasia and Adrenocortical Neoplasia.

CONTEXT: The WNT/ß-catenin pathway is central to the pathogenesis of various human diseases including those affecting bone development and tumor progression. OBJECTIVE: To evaluate the role of a gain-of-function variant in CTNNB1 in a child with a sclerosing bone dysplasia and an adrenocortical adenoma. DESIGN: Whole exome sequencing with corroborative biochemical analyses. PATIENTS: We recruited a child with a sclerosing bone dysplasia and an adrenocortical adenoma together with her unaffected parents. INTERVENTION: Whole exome sequencing and performance of immunoblotting and luciferase-based assays to assess the cellular consequences of a de novo variant in CTNNB1. MAIN OUTCOME MEASURE(S)/RESULT: A de novo variant in CTNNB1 (c.131C>T; p.[Pro44Leu]) was identified in a patient with a sclerosing bone dysplasia and an adrenocortical adenoma. A luciferase-based transcriptional assay of WNT signaling activity verified that the activity of ß-catenin was increased in the cells transfected with a CTNNB1p.Pro44Leu construct (P = 4.00 × 10-5). The ß-catenin p.Pro44Leu variant was also associated with a decrease in phosphorylation at Ser45 and Ser33/Ser37/Thr41 in comparison to a wild-type (WT) CTNNB1 construct (P = 2.16 × 10-3, P = 9.34 × 10-8 respectively). CONCLUSION: Increased ß-catenin activity associated with a de novo gain-of-function CTNNB1 variant is associated with osteosclerotic phenotype and adrenocortical neoplasia.

Biallelic variants in EFEMP1 in a man with a pronounced connective tissue phenotype.

Connective tissue disorders are a spectrum of diseases that affect the integrity of tissues including skin, vasculature, and joints. They are often caused by variants that disrupt genes encoding components of extracellular matrix (ECM). The fibulin glycoproteins are ECM proteins important for integrity of tissues including dermis, retina, fascia, and vasculature. The fibulin family consists of seven members (fibulins-1 to -7) and is defined by a fibulin-type domain at the C-terminus. The family is associated with human diseases, for instance a variant in FBLN1, encoding fibulin-1, is associated with synpolydactyly, while one in EFEMP1, encoding fibulin-3, causes Doyne honeycomb degeneration of the retina. Loss-of-function of fibulins-4 and -5 causes cutis laxa, while variants in fibulins-5 and -6 are associated with age-related macular degeneration. Of note, EFEMP1 is not currently associated with any connective tissue disorder. Here we show biallelic loss-of-function variants in EFEMP1 in an individual with multiple and recurrent abdominal and thoracic herniae, myopia, hypermobile joints, scoliosis, and thin translucent skin. Fibroblasts from this individual express significantly lower EFEMP1 transcript than age-matched control cells. A skin biopsy, visualised using light microscopy, showed normal structure and abundance of elastic fibres. The phenotype of this individual is remarkably similar to the Efemp1 knockout mouse model that displays multiple herniae with premature aging and scoliosis. We conclude that loss of EFEMP1 function in this individual is the cause of a connective tissue disorder with a novel combination of phenotypic features, and can perhaps explain similar, previously reported cases in the literature.

Missense Pathogenic variants in KIF4A Affect Dental Morphogenesis Resulting in X-linked Taurodontism, Microdontia and Dens-Invaginatus.

The etiology of dental anomalies is multifactorial; and genetic and environmental factors that affect the dental lamina have been implicated. We investigated two families of European ancestry in which males were affected by taurodontism, microdontia and dens invaginatus. In both families, males were related to each other via unaffected females. A linkage analysis was conducted in a New Zealand family, followed by exome sequencing and focused analysis of the X-chromosome. In a US family, exome sequencing of the X-chromosome was followed by Sanger sequencing to conduct segregation analyses. We identified two independent missense variants in KIF4A that segregate in affected males and female carriers. The variant in a New Zealand family (p.Asp371His) predicts the substitution of a residue in the motor domain of the protein while the one in a US family (p.Arg771Lys) predicts the substitution of a residue in the domain that interacts with Protein Regulator of Cytokinesis 1 (PRC1). We demonstrated that the gene is expressed in the developing tooth bud during development, and that the p.Arg771Lys variant influences cell migration in an in vitro assay. These data implicate missense variations in KIF4A in a pathogenic mechanism that causes taurodontism, microdontia and dens invaginatus phenotypes.

A Primate-Specific Isoform of PLEKHG6 Regulates Neurogenesis and Neuronal Migration.

The mammalian neocortex has undergone remarkable changes through evolution. A consequence of such rapid evolutionary events could be a trade-off that has rendered the brain susceptible to certain neurodevelopmental and neuropsychiatric conditions. We analyzed the exomes of 65 patients with the structural brain malformation periventricular nodular heterotopia (PH). De novo coding variants were observed in excess in genes defining a transcriptomic signature of basal radial glia, a cell type linked to brain evolution. In addition, we located two variants in human isoforms of two genes that have no ortholog in mice. Modulating the levels of one of these isoforms for the gene PLEKHG6 demonstrated its role in regulating neuroprogenitor differentiation and neuronal migration via RhoA, with phenotypic recapitulation of PH in human cerebral organoids. This suggests that this PLEKHG6 isoform is an example of a primate-specific genomic element supporting brain development.

Genetic investigation into an increased susceptibility to biliary atresia in an extended New Zealand Maori family.

BACKGROUND: Biliary atresia (BA), a fibrosing disorder of the developing biliary tract leading to liver failure in infancy, has an elevated incidence in indigenous New Zealand (NZ) Maori. We investigated a high rate of BA in a group of children (n = 12) belonging to a single Maori iwi (or 'tribe', related through a remote ancestor). METHODS: Population and geographical data was used to estimate the rate of BA in Maori sub-groups, and a pedigree linking most of the affected children was constructed from oral and documented history. Array genotyping was used to examine hypotheses about the inheritance of a possible genetic risk factor, and the history of the affected population, and Exome Sequencing to search for candidate genes. RESULTS: Most of these affected children (n = 7) link to a self-reported pedigree and carry a 50-fold increase in BA risk over unrelated Maori (χ2 = 296P < 0.001, 95% CI 23-111). Genetic analysis using FEstim and SNP array genotypes revealed no evidence for elevated consanguinity between parents of affected children (FEstim: F (2,21) = 0.469, P > 0.63). Genome-wide quantitation of intervals of contiguous, homozygous-by-state markers reached a similar conclusion (F (2,399) = 1.99, P = 0.138). Principal component analysis and investigation with STRUCTURE found no evidence of increased allele frequency of either a recessive variant, or additive, low-risk variants due to reproductive isolation. To identify candidate causal factors, Exome Sequencing datasets were scrutinised for shared rare coding variants across 8 affected individuals. No rare, non-synonymous, phylogenetically conserved variants were common to 6 or more affected children. CONCLUSION: The substantially elevated risk for development of BA in this subgroup could be mediated by genetic factors, but the iwi exhibits no properties indicative of recent or remote reproductive isolation. Resolution of any risk loci may rely on extensive genomic sequencing studies in this iwi or investigation of other mechnaisms such as copy number variation.

Germline mutations and somatic inactivation of TRIM28 in Wilms tumour.

Wilms tumour is a childhood tumour that arises as a consequence of somatic and rare germline mutations, the characterisation of which has refined our understanding of nephrogenesis and carcinogenesis. Here we report that germline loss of function mutations in TRIM28 predispose children to Wilms tumour. Loss of function of this transcriptional co-repressor, which has a role in nephrogenesis, has not previously been associated with cancer. Inactivation of TRIM28, either germline or somatic, occurred through inactivating mutations, loss of heterozygosity or epigenetic silencing. TRIM28-mutated tumours had a monomorphic epithelial histology that is uncommon for Wilms tumour. Critically, these tumours were negative for TRIM28 immunohistochemical staining whereas the epithelial component in normal tissue and other Wilms tumours stained positively. These data, together with a characteristic gene expression profile, suggest that inactivation of TRIM28 provides the molecular basis for defining a previously described subtype of Wilms tumour, that has early age of onset and excellent prognosis.

Recessive Spondylocarpotarsal Synostosis Syndrome Due to Compound Heterozygosity for Variants in MYH3.

Spondylocarpotarsal synostosis syndrome (SCTS) is characterized by intervertebral fusions and fusion of the carpal and tarsal bones. Biallelic mutations in FLNB cause this condition in some families, whereas monoallelic variants in MYH3, encoding embryonic heavy chain myosin 3, have been implicated in dominantly inherited forms of the disorder. Here, five individuals without FLNB mutations from three families were hypothesized to be affected by recessive SCTS on account of sibling recurrence of the phenotype. Initial whole-exome sequencing (WES) showed that all five were heterozygous for one of two independent splice-site variants in MYH3. Despite evidence indicating that three of the five individuals shared two allelic haplotypes encompassing MYH3, no second variant could be located in the WES datasets. Subsequent genome sequencing of these three individuals demonstrated a variant altering a 5' UTR splice donor site (rs557849165 in MYH3) not represented by exome-capture platforms. When the cohort was expanded to 16 SCTS-affected individuals without FLNB mutations, nine had truncating mutations transmitted by unaffected parents, and six inherited the rs557849165 variant in trans, an observation at odds with the population allele frequency for this variant. The rs557849165 variant disrupts splicing in the 5' UTR but is still permissive of MYH3 translational initiation, albeit with reduced efficiency. Although some MYH3 variants cause dominant SCTS, these data indicate that others (notably truncating variants) do not, except in the context of compound heterozygosity for a second hypomorphic allele. These observations make genetic diagnosis challenging in the context of simplex presentations of the disorder.

Mob2 Insufficiency Disrupts Neuronal Migration in the Developing Cortex.

Disorders of neuronal mispositioning during brain development are phenotypically heterogeneous and their genetic causes remain largely unknown. Here, we report biallelic variants in a Hippo signaling factor-MOB2-in a patient with one such disorder, periventricular nodular heterotopia (PH). Genetic and cellular analysis of both variants confirmed them to be loss-of-function with enhanced sensitivity to transcript degradation via nonsense mediated decay (NMD) or increased protein turnover via the proteasome. Knockdown of Mob2 within the developing mouse cortex demonstrated its role in neuronal positioning. Cilia positioning and number within migrating neurons was also impaired with comparable defects detected following a reduction in levels of an upstream modulator of Mob2 function, Dchs1, a previously identified locus associated with PH. Moreover, reduced Mob2 expression increased phosphorylation of Filamin A, an actin cross-linking protein frequently mutated in cases of this disorder. These results reveal a key role for Mob2 in correct neuronal positioning within the developing cortex and outline a new candidate locus for PH development.

Differential regulation of two FLNA transcripts explains some of the phenotypic heterogeneity in the loss-of-function filaminopathies.

Loss-of-function mutations in the X-linked gene FLNA can lead to abnormal neuronal migration, vascular and cardiac defects, and congenital intestinal pseudo-obstruction (CIPO), the latter characterized by anomalous intestinal smooth muscle layering. Survival in male hemizygotes for such mutations is dependent on retention of residual FLNA function but it is unclear why a subgroup of males with mutations in the 5' end of the gene can present with CIPO alone. Here, we demonstrate evidence for the presence of two FLNA isoforms differing by 28 residues at the N-terminus initiated at ATG+1 and ATG+82 . A male with CIPO (c.18_19del) exclusively expressed FLNA ATG+82 , implicating the longer protein isoform (ATG+1 ) in smooth muscle development. In contrast, mutations leading to reduction of both isoforms are associated with compound phenotypes affecting the brain, heart, and intestine. RNA-seq data revealed three distinct transcription start sites, two of which produce a protein isoform utilizing ATG+1 while the third utilizes ATG+82 . Transcripts sponsoring translational initiation at ATG+1 predominate in intestinal smooth muscle, and are more abundant compared with the level measured in fibroblasts. Together these observations describe a new mechanism of tissue-specific regulation of FLNA that could reflect the differing mechanical requirements of these cell types during development.

Mutations in the netrin-1 gene cause congenital mirror movements.

Netrin-1 is a secreted protein that was first identified 20 years ago as an axon guidance molecule that regulates midline crossing in the CNS. It plays critical roles in various tissues throughout development and is implicated in tumorigenesis and inflammation in adulthood. Despite extensive studies, no inherited human disease has been directly associated with mutations in NTN1, the gene coding for netrin-1. Here, we have identified 3 mutations in exon 7 of NTN1 in 2 unrelated families and 1 sporadic case with isolated congenital mirror movements (CMM), a disorder characterized by involuntary movements of one hand that mirror intentional movements of the opposite hand. Given the diverse roles of netrin-1, the absence of manifestations other than CMM in NTN1 mutation carriers was unexpected. Using multimodal approaches, we discovered that the anatomy of the corticospinal tract (CST) is abnormal in patients with NTN1-mutant CMM. When expressed in HEK293 or stable HeLa cells, the 3 mutated netrin-1 proteins were almost exclusively detected in the intracellular compartment, contrary to WT netrin-1, which is detected in both intracellular and extracellular compartments. Since netrin-1 is a diffusible extracellular cue, the pathophysiology likely involves its loss of function and subsequent disruption of axon guidance, resulting in abnormal decussation of the CST.

WDR26 Haploinsufficiency Causes a Recognizable Syndrome of Intellectual Disability, Seizures, Abnormal Gait, and Distinctive Facial Features.

We report 15 individuals with de novo pathogenic variants in WDR26. Eleven of the individuals carry loss-of-function mutations, and four harbor missense substitutions. These 15 individuals comprise ten females and five males, and all have intellectual disability with delayed speech, a history of febrile and/or non-febrile seizures, and a wide-based, spastic, and/or stiff-legged gait. These subjects share a set of common facial features that include a prominent maxilla and upper lip that readily reveal the upper gingiva, widely spaced teeth, and a broad nasal tip. Together, these features comprise a recognizable facial phenotype. We compared these features with those of chromosome 1q41q42 microdeletion syndrome, which typically contains WDR26, and noted that clinical features are consistent between the two subsets, suggesting that haploinsufficiency of WDR26 contributes to the pathology of 1q41q42 microdeletion syndrome. Consistent with this, WDR26 loss-of-function single-nucleotide mutations identified in these subjects lead to nonsense-mediated decay with subsequent reduction of RNA expression and protein levels. We derived a structural model of WDR26 and note that missense variants identified in these individuals localize to highly conserved residues of this WD-40-repeat-containing protein. Given that WDR26 mutations have been identified in ∼1 in 2,000 of subjects in our clinical cohorts and that WDR26 might be poorly annotated in exome variant-interpretation pipelines, we would anticipate that this disorder could be more common than currently appreciated.

A defect in myoblast fusion underlies Carey-Fineman-Ziter syndrome.

Multinucleate cellular syncytial formation is a hallmark of skeletal muscle differentiation. Myomaker, encoded by Mymk (Tmem8c), is a well-conserved plasma membrane protein required for myoblast fusion to form multinucleated myotubes in mouse, chick, and zebrafish. Here, we report that autosomal recessive mutations in MYMK (OMIM 615345) cause Carey-Fineman-Ziter syndrome in humans (CFZS; OMIM 254940) by reducing but not eliminating MYMK function. We characterize MYMK-CFZS as a congenital myopathy with marked facial weakness and additional clinical and pathologic features that distinguish it from other congenital neuromuscular syndromes. We show that a heterologous cell fusion assay in vitro and allelic complementation experiments in mymk knockdown and mymkinsT/insT zebrafish in vivo can differentiate between MYMK wild type, hypomorphic and null alleles. Collectively, these data establish that MYMK activity is necessary for normal muscle development and maintenance in humans, and expand the spectrum of congenital myopathies to include cell-cell fusion deficits.