ABSTRACT
OBJECTIVES: CLCN4 variations have recently been identified as a genetic cause of X-linked neurodevelopmental disorders. This study aims to broaden the phenotypic spectrum of CLCN4-related condition and correlate it with functional consequences of CLCN4 variants. METHODS: We described 13 individuals with CLCN4-related neurodevelopmental disorder. We analyzed the functional consequence of the unreported variants using heterologous expression, biochemistry, confocal fluorescent microscopy, patch-clamp electrophysiology, and minigene splicing assay. RESULTS: We identified five novel (p.R41W, p.L348V, p.G480R, p.R603W, c.1576 + 5G > A) and three known (p.T203I, p.V275M, p.A555V) pathogenic CLCN4 variants in 13 Chinese patients. The p.V275M variant is found at high frequency and seen in four unrelated individuals. All had global developmental delay (GDD)/intellectual disability (ID). Seizures were present in eight individuals, and 62.5% of them developed refractory epilepsy. Five individuals without seizures showed moderate to severe GDD/ID. Developmental delay precedes seizure onset in most patients. The variants p.R41W, p.L348V, and p.R603W compromise the anion/exchange function of ClC-4. p.R41W partially impairs ClC-3/ClC-4 association. p.G480R reduces ClC-4 expression levels and impairs the heterodimerization with ClC-3. The c.1576 + 5G > A variant causes 22 bp deletion of exon 10. CONCLUSIONS: We further define and broaden the clinical and mutational spectrum of CLCN4-related neurodevelopmental conditions. The p.V275M variant may be a potential hotspot CLCN4 variant in Chinese patients. The five novel variants cause loss of function of ClC-4. Transport dysfunction, protein instability, intracellular trafficking defect, or failure of ClC-4 to oligomerize may contribute to the pathophysiological events leading to CLCN4-related neurodevelopmental disorder.
ABSTRACT
Six bread formulations were developed, using different proportions of whole-wheat flour, chia seeds and flaxseed flour. All of our formulations were added with folic acid. Sensorial and texture evaluations were performed, showing good acceptance of the products. Proximal chemical analysis was carried out; in addition, the following parameters were determined: calcium, phosphorus, total dietary fiber, folic acid, water hydration capacity, Glucose Dialysis Retardation Index (GDRI) and fatty acids. The results obtained showed higher protein levels in the developed breads (23.23-30.24 (g/100g dry matter) as compared to a control (21.00% of proteins in bread elaborated without chia or flaxseed). Furthermore, the breads contained 10.07-12.15 of lipids (g/100g dry matter) (linoleic acid: 2.43-4.05%; linolenic acid: 1.12-4.46 %; oleic acid: 2.93-6.13 %), GDRI values were between 89.1 and 98.1 % and folic acid was in the range 699.44 - 991.3 (microg/100g dry matter). The same parameters were determined in the chia seed and in the flaxseed flour. It was concluded that; due to their high levels of protein, insaturated fatty acids (omega-3 and omega-6), dietary fiber and folic acid, these breads have a high nutritional value, so they could have special benefits for woman.
