Novel USH1G homozygous variant underlying USH2-like phenotype of Usher syndrome

PURPOSE: Usher syndrome (USH) is an autosomal recessive disorder characterized by congenital sensorineural hearing impairment and retinitis pigmentosa. Classification distinguishes three clinical types of which type I (USH1) is the most severe, with vestibular dysfunction as an added feature. To date, 15 genes and 3 loci have been identified with the USH1G gene being an uncommon cause of USH. We describe an atypical USH1G-related phenotype caused by a novel homozygous missense variation in a patient with profound hearing impairment and relatively mild retinitis pigmentosa, but no vestibular dysfunction. METHODS: A 26-year-old female patient with profound congenital sensorineural hearing loss, nyctalopia and retinitis pigmentosa was studied. Audiometric, vestibular and ophthalmologic examination was performed. A panel of 13 genes was tested by next-generation sequencing (NGS). RESULTS: While the hearing loss was confirmed to be profound, the vestibular function resulted normal. Although typical retinitis pigmentosa was present, the age at onset was unusually late for USH1 syndrome. A novel homozygous missense variation (c.1187T>A, p.Leu396Gln) in the USH1G gene has been identified as causing the disease in our patient. CONCLUSIONS: Genetic and phenotypic heterogeneity are very common in both isolated and syndromic retinal dystrophies and sensorineural hearing loss. Our findings widen the spectrum of USH allelic disorders and strength the concept that variants in genes that are classically known as underlying one specific clinical USH subtype might result in unexpected phenotypes

Foxc2 disease mutations identified in lymphedema distichiasis patients impair transcriptional activity and cell proliferation

FOXC2 is a member of the human forkhead-box gene family and encodes a regulatory transcription factor. Mutations in FOXC2 have been associated with lymphedema distichiasis (LD), an autosomal dominant disorder that primarily affects the limbs. Most patients also show extra eyelashes, a condition known as distichiasis. We previously reported genetic and clinical findings in six unrelated families with LD. Half the patients showed missense mutations, two carried frameshift mutations and a stop mutation was identified in a last patient. Here we analyzed the subcellular localization and transactivation activity of the mutant proteins, showing that all but one (p.Y109*) localized to the nucleus. A significant reduction of transactivation activity was observed in four mutants (p.L80F, p.H199Pfs*264, p.I213Tfs*18, p.Y109*) compared with wild type FOXC2 protein, while only a partial loss of function was associated with p.V228M. The mutant p.I213V showed a very slight increase of transactivation activity. Finally, immunofluorescence analysis revealed that some mutants were sequestered into nuclear aggregates and caused a reduction of cell viability. This study offers new insights into the effect of FOXC2 mutations on protein function and shows the involvement of aberrant aggregation of FOXC2 proteins in cell death

Imbalance between Expression of FOXC2 and Its lncRNA in Lymphedema-Distichiasis Caused by Frameshift Mutations

Forkhead-box C2 (FOXC2) is a transcription factor involved in lymphatic system development. FOXC2 mutations cause Lymphedema-distichiasis syndrome (LD). Recently, a natural antisense was identified, called lncRNA FOXC2-AS1, which increases FOXC2 mRNA stability. No studies have evaluated FOXC2 and FOXC2-AS1 blood expression in LD and healthy subjects. Here, we show that FOXC2 and FOXC-AS1 expression levels were similar in both controls and patients, and a significantly higher amount of both RNAs was observed in females. A positive correlation between FOXC2 and FOXC2-AS1 expression was found in both controls and patients, excluding those with frameshift mutations. In these patients, the FOXC2-AS1/FOXC2 ratio was about 1:1, while it was higher in controls and patients carrying other types of mutations. The overexpression or silencing of FOXC2-AS1 determined a significant increase or reduction in FOXC2 wild-type and frameshift mutant proteins, respectively. Moreover, confocal and bioinformatic analysis revealed that these variations caused the formation of nuclear proteins aggregates also involving DNA. In conclusion, patients with frameshift mutations presented lower values of the FOXC2-AS1/FOXC2 ratio, due to a decrease in FOXC2-AS1 expression. The imbalance between FOXC2 mRNA and its lncRNA could represent a molecular mechanism to reduce the amount of FOXC2 misfolded proteins, protecting cells from damage

Genetic evaluation of AMPD1, CPT2, and PGYM metabolic enzymes in patients with chronic fatigue syndrome

Chronic fatigue syndrome (CFS) is a disease that can seriously impair one’s quality of life; patients complain of excessive fatigue and myalgia following physical exertion. This disease may be associated with abnormalities in genes affecting exercise tolerance and physical performance. Adenosine monophosphate deaminase (AMPD1), carnitine palmitoyltransferase II (CPT2), and the muscle isoform of glycogen phosphorylase (PYGM) genes provide instructions for producing enzymes that play major roles in energy production during work. The aim of this study was to look for evidence of genotypeassociated excessive muscle fatigue. Three metabolic genes (AMPD1, CPT2, and PYGM) were therefore fully sequenced in 17 Italian patients with CFS. We examined polymorphisms known to alter the function of these metabolic genes, and compared their genotypic distributions in CFS patients and 50 healthy controls using chi-square tests and odds ratios. One-way analysis of variance with F-ratio was carried out to determine the associations between genotypes and disease severity using CF scores. No major genetic variations between patients and controls were found in the three genes studied, and we did not find any association between these genes and CFS. In conclusion, variations in AMPD1, CPT2, and PGYM genes are not associated with the onset, susceptibility, or severity of CFS

FOXC2 disease-mutations identified in lymphedema-distichiasis patients cause both loss and gain of protein function

Dominant mutations in the FOXC2 gene cause a form of lymphedema primarily of the limbs that usually develops at or after puberty. In 90-95% of patients, lymphedema is accompanied by distichiasis. FOXC2 is a member of the forkhead/winged-helix family of transcription factors and plays essential roles in different developmental pathways and physiological processes. We previously described six unrelated families with primary lymphedema-distichiasis in which patients showed different FOXC2 mutations located outside of the forkhead domain. Of those, four were missense mutations, one a frameshift mutation, and the last a stop mutation. To assess their pathogenic potential, we have now examined the subcellular localization and the transactivation activity of the mutated FOXC2 proteins. All six FOXC2 mutant proteins were able to localize into the nucleus; however, the frameshift truncated protein appeared to be sequestered into nuclear aggregates. A reduction in the ability to activate FOXC1/FOXC2 response elements was detected in 50% of mutations, while the remaining ones caused an increase of protein transactivation activity. Our data reveal that either a complete loss or a significant gain of FOXC2 function can cause a perturbation of lymphatic vessel formation leading to lymphedema

Prevalence of mutations in LEP, LEPR, and MC4R genes in individuals with severe obesity

Obesity is a major public health concern; despite evidence of high heritability, the genetic causes of obesity remain unclear. In this study, we assessed the presence of mutations in three genes involved in the hypothalamic leptin-melanocortin regulation pathway (leptin, LEP; leptin receptor, LEPR; and melanocortin-4 receptor, MC4R), which is important for energy homeostasis in the body, in a group of patients with severe obesity. For this study, we selected 77 patients who had undergone bariatric surgery and had a pre-operative body mass index (BMI) >35 kg/m2, early onset and a family history of being overweight. Candidate genes were screened by direct sequence analysis to search for rare genetic variations. The common LEP -2548 G/A polymorphism was also evaluated for its influence on the BMI (in obesity patients) and for obesity risk, using a case-control study involving 117 healthy individuals. Two different non-synonymous alterations in MC4R were found in two patients: the p.(Thr112Met), previously described in the literature as a probable gene involved in the obesity phenotype, and the novel p.(Tyr302Asp) variant, predicted to be pathogenic by in silico evaluations and family segregation studies. The LEP -2548 G/A polymorphism was not associated with the BMI or obesity risk. In conclusion, we have reported a novel mutation in MC4R in a family of Italian patients with severe obesity. Screening for MC4R could be important for directing the carriers of mutations towards therapy including partial agonists of the MC4R that could normalize their appetite and inhibit compulsive eating. Next-generation sequencing could be used to clarify the genetic basis of obesity in the future