ZMYND11-related syndromic intellectual disability: 16 patients delineating and expanding the phenotypic spectrum.

Pathogenic variants in ZMYND11, which acts as a transcriptional repressor, have been associated with intellectual disability, behavioral abnormalities, and seizures. Only 11 affected individuals have been reported to date, and the phenotype associated with pathogenic variants in this gene have not been fully defined. Here, we present 16 additional patients with predicted pathogenic heterozygous variants in including four individuals from the same family, to further delineate and expand the genotypic and phenotypic spectrum of ZMYND11-related syndromic intellectual disability. The associated phenotype includes developmental delay, particularly affecting speech, mild-moderate intellectual disability, significant behavioral abnormalities, seizures, and hypotonia. There are subtle shared dysmorphic features, including prominent eyelashes and eyebrows, a depressed nasal bridge with bulbous nasal tip, anteverted nares, thin vermilion of the upper lip, and wide mouth. Novel features include brachydactyly and tooth enamel hypoplasia. Most identified variants are likely to result in premature truncation and/or nonsense-mediated decay. Two ZMYND11 variants located in the final exon-p.(Gln586*) (likely escaping nonsense-mediated decay) and p.(Cys574Arg)-are predicted to disrupt the MYND-type zinc-finger motif and likely interfere with binding to its interaction partners. Hence, the homogeneous phenotype likely results from a common mechanism of loss-of-function.

Identification of a mutation in CNNM4 by whole exome sequencing in an Amish family and functional link between CNNM4 and IQCB1.

We investigated an Amish family in which three siblings presented with an early-onset childhood retinal dystrophy inherited in an autosomal recessive fashion. Genome-wide linkage analysis identified significant linkage to marker D2S2216 on 2q11 with a two-point LOD score of 1.95 and a multi-point LOD score of 3.76. Whole exome sequencing was then performed for the three affected individuals and identified a homozygous nonsense mutation (c.C1813T, p.R605X) in the cyclin and CBS domain divalent metal cation transport mediator 4 (CNNM4) gene located within the 2p14-2q14 Jalili syndrome locus. The initial assessment and collection of the family were performed before the clinical delineation of Jalili syndrome. Another assessment was made after the discovery of the responsible gene and the dental abnormalities characteristic of Jalili syndrome were retrospectively identified. The p.R605X mutation represents the first probable founder mutation of Jalili syndrome identified in the Amish community. The molecular mechanism underlying Jalili syndrome is unknown. Here we show that CNNM4 interacts with IQCB1, which causes Leber congenital amaurosis (LCA) when mutated. A truncated CNNM4 protein starting at R605 significantly increased the rate of apoptosis, and significantly increased the interaction between CNNM4 and IQCB1. Mutation p.R605X may cause Jalili syndrome by a nonsense-mediated decay mechanism, affecting the function of IQCB1 and apoptosis, or both. Our data, for the first time, functionally link Jalili syndrome gene CNNM4 to LCA gene IQCB1, providing important insights into the molecular pathogenic mechanism of retinal dystrophy in Jalili syndrome.

A nonsense mutation in DHTKD1 causes Charcot-Marie-Tooth disease type 2 in a large Chinese pedigree.

Charcot-Marie-Tooth (CMT) disease represents a clinically and genetically heterogeneous group of inherited neuropathies. Here, we report a five-generation family of eight affected individuals with CMT disease type 2, CMT2. Genome-wide linkage analysis showed that the disease phenotype is closely linked to chromosomal region 10p13-14, which spans 5.41 Mb between D10S585 and D10S1477. DNA-sequencing analysis revealed a nonsense mutation, c.1455T>G (p.Tyr485(∗)), in exon 8 of dehydrogenase E1 and transketolase domain-containing 1 (DHTKD1) in all eight affected individuals, but not in other unaffected individuals in this family or in 250 unrelated normal persons. DHTKD1 mRNA expression levels in peripheral blood of affected persons were observed to be half of those in unaffected individuals. In vitro studies have shown that, compared to wild-type mRNA and DHTKD1, mutant mRNA and truncated DHTKD1 are significantly decreased by rapid mRNA decay in transfected cells. Inhibition of nonsense-mediated mRNA decay by UPF1 silencing effectively rescued the decreased levels of mutant mRNA and protein. More importantly, DHTKD1 silencing was found to lead to impaired energy production, evidenced by decreased ATP, total NAD(+) and NADH, and NADH levels. In conclusion, our data demonstrate that the heterozygous nonsense mutation in DHTKD1 is one of CMT2-causative genetic alterations, implicating an important role for DHTKD1 in mitochondrial energy production and neurological development.

Oculofaciocardiodental syndrome: novel BCOR mutations and expression in dental cells.

Oculofaciocardiodental (OFCD) syndrome is a rare X-linked dominant condition. Mutations in BCOR have been described as causal in OFCD syndrome. Almost all BCOR mutations result in premature termination codons (PTCs); therefore, nonsense-mediated mRNA decay (NMD) might have an important role in pathogenesis. The purpose of this study was to identify BCOR mutations in two OFCD patients, if it present, and to clarify the pathogenesis of radiculomegaly using one OFCD patient's pulp and periodontal ligament (PDL) cells. In our genetic analysis, two novel BCOR mutations were found. We also examined the transcript levels and the effects of NMD using cultured pulp and PDL cells from one affected patient. BCOR expression was normal in pulp but reduced in PDL cells, which is consistent with the higher rates of NMD in PDL cells. The mutant PDL cells had unstable mutant transcripts and proliferated faster than did wild-type cells, but mutant pulp cells appeared normal by these measures. In summary, the nonsense and frameshift mutations, which introduce PTCs, were found to contribute to OFCD syndrome in our two patients. Furthermore, in PDL cells, the mutation resulting in a PTC corresponded to greater NMD, unstable mutant transcripts and increased cell proliferation, which may contribute to hyperactive root formation.

Nonsense-mediated mRNA decay affects hyperactive root formation in oculo-facio-cardio-dental syndrome via up-frameshift protein 1.

OBJECTIVES: Oculo-facio-cardio-dental (OFCD) syndrome is a rare X-linked genetic disorder caused by mutations in the BCL6 co-repressor (BCOR) and is mainly characterized by radiculomegaly (elongated dental roots). All BCOR mutations reported to date have been associated with premature termination codons, indicating that nonsense-mediated mRNA decay (NMD) might play a vital role in the pathogenesis of OFCD syndrome. However, the molecular mechanisms underlying NMD remain unclear. In this study, we investigated the involvement of up-frameshift protein 1 (UPF1), which plays a central role in NMD, in the hyperactive root formation caused by BCOR mutations. METHODS: Periodontal ligament cells, isolated from a Japanese woman with a c.3668delC frameshift mutation in BCOR, and primary human periodontal ligament fibroblasts (HPdLFs) were used for an RNA immunoprecipitation assay to confirm the binding of UPF1 to mutated BCOR. Additionally, the effects of UPF1 on the BCOR transcription levels and corresponding gene expression were determined by performing relative quantitative real-time polymerase chain reactions. RESULTS: RNA immunoprecipitation revealed that UPF1 binds to exon 9 of mutated BCOR. Additionally, UPF1 knockdown via siRNA upregulated the transcription of BCOR, whereas overexpression of wild-type and mutated BCOR with the same frameshift mutation in HPdLFs altered bone morphogenetic protein 2 (BMP2) expression. CONCLUSIONS: Our findings indicate that BCOR mutations regulate the transcription of BCOR via UPF1, which may in turn regulate the expression of BMP2. NMD, caused by a c.3668delC mutation, potentially leads to an OFCD syndrome phenotype, including elongated dental roots.

Identification of a Novel COL17A1 Compound Heterozygous Mutation in a Chinese Girl with Non-Herlitz Junctional Epidermolysis Bullosa.

Non-Herlitz junctional epidermolysis bullosa (JEB-nH), an autosomal recessive bullous genodermatosis, is characterized by generalized skin blistering from birth onward, dental anomalies, universal alopecia and nail dystrophy. The underlying defect is mutation of the COL17A1 gene encoding the type XVII collagen, resulting in losing structure for attachment of basal epithelial cells to the matrix. In present study, we described one case of congenitally affected female child aged 10 years, with skin blistering. Dermatologic examination revealed sparse, mild blisters on the face and hand, with profound enamel pitting of the teeth. Skin biopsy from proband's bullous skin displayed subepidermal bulla formation without acantholysis. The immunofluorescence of anti-type XVII collagen antibody staining showed loss of type XVII collagen staining at the basement membrane zone. A combination of whole exome sequencing (WES) and Sanger sequencing revealed the novel heterozygous mutations (c.4324C>T;p.Q1442* and c.1834G>C;p.G612R) in COL17A1 gene, which could be associated with the observed JEB-nH. One allele had a novel nonsense mutation (c.4324C>T;p.Q1442*), resulting in nonsense-mediated mRNA decay and truncated collagen XVII; the other allele had a novel missense mutation of c.1834G>C;p.G612R in exon 22, causing a glycine-to-arginine substitution in the Gly-X-Y triple helical repeating motifs and decreasing the thermal stability of collagen XVII. Our findings indicate that the genetic test based on WES can be useful in diagnosing JEB-nH patients. The novel pathogenic mutations identified would further expand our understanding of the mutation spectrum of COL17A1 gene in association with the inherited blistering diseases.

CMT2Q-causing mutation in the Dhtkd1 gene lead to sensory defects, mitochondrial accumulation and altered metabolism in a knock-in mouse model.

Charcot-Marie-Tooth disease (CMT) is a group of inherited neurological disorders of the peripheral nervous system. CMT is subdivided into two main types: a demyelinating form, known as CMT1, and an axonal form, known as CMT2. Nearly 30 genes have been identified as a cause of CMT2. One of these is the 'dehydrogenase E1 and transketolase domain containing 1' (DHTKD1) gene. We previously demonstrated that a nonsense mutation [c.1455 T > G (p.Y485*)] in exon 8 of DHTKD1 is one of the disease-causing mutations in CMT2Q (MIM 615025). The aim of the current study was to investigate whether human disease-causing mutations in the Dhtkd1 gene cause CMT2Q phenotypes in a mouse model in order to investigate the physiological function and pathogenic mechanisms associated with mutations in the Dhtkd1 gene in vivo. Therefore, we generated a knock-in mouse model with the Dhtkd1Y486* point mutation. We observed that the Dhtkd1 expression level in sciatic nerve of knock-in mice was significantly lower than in wild-type mice. Moreover, a histopathological phenotype was observed, reminiscent of a peripheral neuropathy, including reduced large axon diameter and abnormal myelination in peripheral nerves. The knock-in mice also displayed clear sensory defects, while no abnormalities in the motor performance were observed. In addition, accumulation of mitochondria and an elevated energy metabolic state was observed in the knock-in mice. Taken together, our study indicates that the Dhtkd1Y486* knock-in mice partially recapitulate the clinical phenotypes of CMT2Q patients and we hypothesize that there might be a compensatory effect from the elevated metabolic state in the knock-in mice that enables them to maintain their normal locomotor function.

Whole genome sequencing reveals novel IGHMBP2 variant leading to unique cryptic splice-site and Charcot-Marie-Tooth phenotype with early onset symptoms.

BACKGROUND: Rare variants (RV) in immunoglobulin mu-binding protein 2 (IGHMBP2) [OMIM 600502] can cause an autosomal recessive type of Charcot-Marie-Tooth (CMT) disease [OMIM 616155], an inherited peripheral neuropathy. Over 40 different genes are associated with CMT, with different possible inheritance patterns. METHODS AND RESULTS: An 11-year-old female with motor delays was found to have distal atrophy, weakness, and areflexia without bulbar or sensory findings. Her clinical evaluation was unrevealing. Whole exome sequencing (WES) revealed a maternally inherited IGHMBP2 RV (c.1730T>C) predicted to be pathogenic, but no variant on the other allele was identified. Deletion and duplication analysis was negative. She was referred to the Undiagnosed Disease Network (UDN) for further evaluation. Whole genome sequencing (WGS) confirmed the previously identified IGHMBP2 RV and identified a paternally inherited non-coding IGHMBP2 RV. This was predicted to activate a cryptic splice site perturbing IGHMBP2 splicing. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis was consistent with activation of the cryptic splice site. The abnormal transcript was shown to undergo nonsense-mediated decay (NMD), resulting in halpoinsufficiency. CONCLUSION: This case demonstrates the deficiencies of WES and traditional molecular analyses and highlights the advantages of utilization of WGS and functional studies.