Expanding the phenotypic spectrum of CC2D2A-related ciliopathies: a rare homozygous nonsense variant in a patient with suspected nephronophthisis.

Biallelic pathogenic variants in the gene CC2D2A cause a spectrum of ciliopathies, including Joubert and Meckel syndrome, which frequently involve the kidney; however, no cases of isolated renal disease (i.e., nephronophthisis) have yet been reported. In an adult with a clinical presentation consistent with nephronophthisis, next-generation sequencing identified a rare homozygous nonsense variant in CC2D2A (c.100 C > T; p.(Arg34*)). Tissue-specific expression data and promoter activity analysis demonstrates that this variant primarily affects a transcript isoform predominant in the kidneys but does not affect the transcript isoforms predominant in other tissues typically involved in CC2D2A-related ciliopathies (e.g., cerebellum, liver). Expression analysis of patient-specific cDNA in MDCK cells demonstrates partial translation re-initiation downstream of p.(Arg34*) as a possible escape mechanism from nonsense mediated decay. These data provide mechanistic insights in support of this novel genotype-phenotype association.

Mutations in SCARF2 are responsible for Van Den Ende-Gupta syndrome.

Van Den Ende-Gupta syndrome (VDEGS) is an extremely rare autosomal-recessive disorder characterized by distinctive craniofacial features, which include blepharophimosis, malar and/or maxillary hypoplasia, a narrow and beaked nose, and an everted lower lip. Other features are arachnodactyly, camptodactyly, peculiar skeletal abnormalities, and normal development and intelligence. We present molecular data on four VDEGS patients from three consanguineous Qatari families belonging to the same highly inbred Bedouin tribe. The patients were genotyped with SNP microarrays, and a 2.4 Mb homozygous region was found on chromosome 22q11 in an area overlapping the DiGeorge critical region. This region contained 44 genes, including SCARF2, a gene that is expressed during development in a number of mouse tissues relevant to the symptoms described above. Sanger sequencing identified a missense change, c.773G>A (p.C258Y), in exon 4 in the two closely related patients and a 2 bp deletion in exon 8, c.1328_1329delTG (p.V443DfsX83), in two unrelated individuals. In parallel with the candidate gene approach, complete exome sequencing was used to confirm that SCARF2 was the gene responsible for VDEGS. SCARF2 contains putative epidermal growth factor-like domains in its extracellular domain, along with a number of positively charged residues in its intracellular domain, indicating that it may be involved in intracellular signaling. However, the function of SCARF2 has not been characterized, and this study reports that phenotypic effects can be associated with defects in the scavenger receptor F family of genes.

Association of novel mutation in TRPV4 with familial nonsyndromic craniosynostosis with complete penetrance and variable expressivity.

OBJECTIVE: The aim of this study was to characterize a novel pathogenic variant in the transient receptor potential vanilloid 4 (TRPV4) gene, causing familial nonsyndromic craniosynostosis (CS) with complete penetrance and variable expressivity. METHODS: Whole-exome sequencing was performed on germline DNA of a family with nonsyndromic CS to a mean depth coverage of 300× per sample, with greater than 98% of the targeted region covered at least 25×. In this study, the authors detected a novel variant, c.496C>A in TRPV4, exclusively in the four affected family members. The variant was modeled using the structure of the TRPV4 protein from Xenopus tropicalis. In vitro assays in HEK293 cells overexpressing wild-type TRPV4 or TRPV4 p.Leu166Met were used to assess the effect of the mutation on channel activity and downstream MAPK signaling. RESULTS: The authors identified a novel, highly penetrant heterozygous variant in TRPV4 (NM_021625.4:c.496C>A) causing nonsyndromic CS in a mother and all three of her children. This variant results in an amino acid change (p.Leu166Met) in the intracellular ankyrin repeat domain distant from the Ca2+-dependent membrane channel domain. In contrast to other TRPV4 mutations in channelopathies, this variant does not interfere with channel activity as identified by in silico modeling and in vitro overexpression assays in HEK293 cells. CONCLUSIONS: Based on these findings, the authors hypothesized that this novel variant causes CS by modulating the binding of allosteric regulatory factors to TRPV4 rather than directly modifying its channel activity. Overall, this study expands the genetic and functional spectrum of TRPV4 channelopathies and is particularly relevant for the genetic counseling of CS patients.

Combined malonic and methylmalonic aciduria: exome sequencing reveals mutations in the ACSF3 gene in patients with a non-classic phenotype.

BACKGROUND: Combined Malonic and Methylmalonic Aciduria (CMAMMA) is a rare recessive inborn error of metabolism characterised by elevations of urine malonic acid (MA) and methylmalonic acid (MMA). Nearly all reported cases are caused by malonyl-CoA decarboxylase (MCD) deficiency. Most patients have metabolic acidosis, developmental delay, seizures and cardiomyopathy. CMAMMA was also described in symptomatic patients with normal MCD activity, suggesting heterogeneity in this disorder. METHODS AND RESULTS: We identified two probands with a non-classical CMAMMA variant through the Quebec newborn urine screening program. While they share the biochemical phenotype of elevated MA and MMA, the MMA excretion was higher than MA, the clinical courses were benign, MYLCD gene sequencing was normal and MCD activity, measured in one proband, was normal. Using exome sequencing in the single consanguineous proband, we identified a homozygous missense allele in the ACSF3 gene, encoding an Acyl-CoA Synthetase (ACS) with unknown substrate and function. The second proband was homozygous for a different ACSF3 missense allele. Both substitutions were in conserved residues and were identified in less than 0.5% of their respective ethnic control populations. CONCLUSION: These results suggest that ACSF3 is a candidate gene for non-classical CMAMMA observed in our patients and document the value of exome sequencing of a limited number of patients for the identification of novel disease genes.

Positive newborn screen for methylmalonic aciduria identifies the first mutation in TCblR/CD320, the gene for cellular uptake of transcobalamin-bound vitamin B(12).

Elevated methylmalonic acid in five asymptomatic newborns whose fibroblasts showed decreased uptake of transcobalamin-bound cobalamin (holo-TC), suggested a defect in the cellular uptake of cobalamin. Analysis of TCblR/CD320, the gene for the receptor for cellular uptake of holo-TC, identified a homozygous single codon deletion, c.262_264GAG (p.E88del), resulting in the loss of a glutamic acid residue in the low-density lipoprotein receptor type A-like domain. Inserting the codon by site-directed mutagenesis fully restored TCblR function.

Spectrum of mutations in MMACHC, allelic expression, and evidence for genotype-phenotype correlations.

Methylmalonic aciduria and homocystinuria, cblC type, is a rare disorder of intracellular vitamin B(12) (cobalamin [Cbl]) metabolism caused by mutations in the MMACHC gene. MMACHC was sequenced from the gDNA of 118 cblC individuals. Eleven novel mutations were identified, as well as 23 mutations that were observed previously. Six sequence variants capture haplotype diversity in individuals across the MMACHC interval. Genotype-phenotype correlations of common mutations were apparent; individuals with c.394C>T tend to present with late-onset disease whereas patients with c.331C>T and c.271dupA tend to present in infancy. Other missense variants were also associated with late- or early-onset disease. Allelic expression analysis was carried out on human cblC fibroblasts compound heterozygous for different combinations of mutations including c.271dupA, c.331C>T, c.394C>T, and c.482G>A. The early-onset c.271dupA mutation was consistently underexpressed when compared to control alleles and the late-onset c.394C>T and c.482G>A mutations. The early-onset c.331C>T mutation was also underexpressed when compared to control alleles and the c.394C>T mutation. Levels of MMACHC mRNA transcript in cell lines homozygous for c.271dupA, c.331C>T, and c.394C>T were assessed using quantitative real-time RT-PCR. Cell lines homozygous for the late onset c.394C>T mutation had significantly higher levels of transcript when compared to cell lines homozygous for the early-onset mutations. Differential or preferential MMACHC transcript levels may provide a clue as to why individuals carrying c.394C>T generally present later in life.

Epigenetic modification of the gene for the vitamin B(12) chaperone MMACHC can result in increased tumorigenicity and methionine dependence.

Methionine dependence, the inability of cells to grow when the amino acid methionine is replaced in culture medium by its metabolic precursor homocysteine, is characteristic of many cancer cell lines and some tumors in situ. Most cell lines proliferate normally under these conditions. The methionine dependent tumorigenic human melanoma cell line MeWo-LC1 was derived from the methionine independent non-tumorigenic line, MeWo. MeWo-LC1 has a cellular phenotype identical to that of cells from patients with the cblC inborn error of cobalamin metabolism, with decreased synthesis of cobalamin coenzymes and decreased activity of the cobalamin-dependent enzymes methionine synthase and methylmalonylCoA mutase. Inability of cblC cells to complement the defect in MeWo-LC1 suggested that it was caused by decreased activity of the MMACHC gene. However, no potentially disease causing mutations were detected in the coding sequence of MMACHC in MeWo-LC1. No MMACHC expression was detected in MeWo-LC1 by quantitative or non-quantitative PCR. There was virtually complete methylation of a CpG island at the 5'-end of the MMACHC gene in MeWo-LC1, consistent with inactivation of the gene by methylation. The CpG island was partially methylated (30-45%) in MeWo and only lightly methylated (2-11%) in control fibroblasts. Infection of MeWo-LC1 with wild type MMACHC resulted in correction of the defect in cobalamin metabolism and restoration of the ability of cells to grow in medium containing homocysteine. We conclude that epigenetic inactivation of the MMACHC gene is responsible for methionine dependence in MeWo-LC1.

Mitochondrial Complex III Deficiency with Ketoacidosis and Hyperglycemia Mimicking Neonatal Diabetes.

Hyperglycemia is a rare presenting symptom of mitochondrial disorders. We report a case of a young girl who presented shortly after birth with ketoacidosis, hyperlactatemia, hyperammonemia, and insulin-responsive hyperglycemia. Initial metabolic work-up suggested mitochondrial dysfunction. Given our patient's unusual presentation, whole-exome sequencing (WES) was performed on the parent-offspring trio. The patient was homozygous for the c.643C>T (p.Leu215Phe) variant in CYC1, a nuclear gene which encodes cytochrome c 1 , a subunit of respiratory chain complex III. Variants in this gene have only been previously reported in two patients with similar presentation, one of whom carries the same variant as our patient who is also of Sri Lankan origin.Primary complex III deficiencies are rare and its phenotypes can vary significantly, even among patients with the same genotype.