Report of a Novel Homozygous Intragenic DCC Duplication and a Review of Literature of Developmental Split-Brain Syndrome aka Horizontal Gaze Palsy with Progressive Scoliosis-2 with Impaired Intellectual Development Syndrome.

Introduction: Horizontal gaze palsy with progressive scoliosis-2 (HGPPS2, MIM 617542) with impaired intellectual development aka developmental split-brain syndrome is an ultra-rare congenital disorder caused by pathogenic biallelic variants in the deleted in colorectal cancer (DCC) gene. Case Presentation: We report the clinical and genetic characterization of a Syrian patient with a HGPPS2 phenotype and review the previously published cases of HGPPS2. The genetic screening was performed using exome sequencing on Illumina platform. Genetic analysis revealed a novel DCC c.(?_1912)_(2359_?)dup, p.(Ser788Tyrfs*4) variant segregating recessively in the family. This type of variant has not been described previously in the HGPPS2 patients. To date, including the case reported here, three different homozygous pathogenic frameshift variants, one homozygous missense variant, and an intragenic duplication in the DCC gene have been reported in 8 patients with the HGPPS2 syndrome. Conclusion: The analysis of duplications and deletions in the DCC should be included in the routine genetic diagnostic evaluation of patients with suspected HGPPS2. This report expands the knowledge of phenotypic and genotypic spectrum of pathogenic variants causing HGPPS2.

Multisite Evaluation and Validation of a Sensitive Diagnostic and Screening System for Spinal Muscular Atrophy that Reports SMN1 and SMN2 Copy Number, along with Disease Modifier and Gene Duplication Variants.

Spinal muscular atrophy is a severe autosomal recessive disease caused by disruptions in the SMN1 gene. The nearly identical SMN2 gene copy number is associated with disease severity. SMN1 duplication markers, such as c.∗3+80T>G and c.∗211_∗212del, can assess residual carrier risk. An SMN2 disease modifier (c.859G>C) can help inform prognostic outcomes. The emergence of multiple precision gene therapies for spinal muscular atrophy requires accurate and rapid detection of SMN1 and SMN2 copy numbers to enable early treatment and optimal patient outcomes. We developed and evaluated a single-tube PCR/capillary electrophoresis assay system that quantifies SMN1/2 copy numbers and genotypes three additional clinically relevant variants. Analytical validation was performed with human cell lines and whole blood representing varying SMN1/2 copies on four capillary electrophoresis instrument models. In addition, four independent laboratories used the assay to test 468 residual clinical genomic DNA samples. The results were ≥98.3% concordant with consensus SMN1/2 exon 7 copy numbers, determined using multiplex ligation-dependent probe amplification and droplet digital PCR, and were 100% concordant with Sanger sequencing for the three variants. Furthermore, copy number values were 98.6% (SMN1) and 97.1% (SMN2) concordant to each laboratory's own reference results.

OGT and OGA expression in postmenopausal skeletal muscle associates with hormone replacement therapy and muscle cross-sectional area.

Protein glycosylation via O-linked N-acetylglucosaminylation (O-GlcNAcylation) is an important post-translational regulatory mechanism mediated by O-GlcNAc transferase (OGT) and responsive to nutrients and stress. OGT attaches an O-GlcNAc moiety to proteins, while O-GlcNAcase (OGA) catalyzes O-GlcNAc removal. In skeletal muscle of experimental animals, prolonged increase in O-GlcNAcylation associates with age and muscle atrophy. Here we examined the effects of hormone replacement therapy (HRT) and power training (PT) on muscle OGT and OGA gene expression in postmenopausal women generally prone to age-related muscle weakness. In addition, the associations of OGT and OGA gene expressions with muscle phenotype were analyzed. Twenty-seven 50-57-year-old women participated in a yearlong randomized placebo-controlled trial: HRT (n=10), PT (n=8) and control (n=9). OGT and OGA mRNA levels were measured from muscle samples obtained at baseline and after one year. Knee extensor muscle cross-sectional area (CSA), knee extension force, running speed and vertical jumping height were measured. During the yearlong intervention, HRT suppressed the aging-associated upregulation of OGT mRNA that occurred in the controls. The effects of PT were similar but weaker. HRT also tended to increase the OGA mRNA level compared to the controls. The change in the ratio of OGT to OGA gene expressions correlated negatively with the change in muscle CSA. Our results suggest that OGT and OGA gene expressions are associated with muscle size during the critical postmenopausal period. HRT and PT influence muscle OGT and OGA gene expression, which may be one of the mechanisms by which HRT and PT prevent aging-related loss of muscle mass.

Interactions of y+LAT1 and 4F2hc in the y+l amino acid transporter complex: consequences of lysinuric protein intolerance-causing mutations.

Lysinuric protein intolerance (LPI) is an inherited aminoaciduria caused by recessive mutations in the SLC7A7 gene encoding y+L amino acid transporter 1 (y+LAT1), which combines with 4F2hc to generate an active transporter responsible for the system y+L amino acid transport. We have previously shown that the y+LAT1 proteins with point mutations are expressed in the plasma membrane, while those with frameshift mutations are retained in the cytoplasm. This finding has prompted us to study whether the difference in localization is due to the inability of the structurally altered mutant y+LAT1 proteins to heteromerize with 4F2hc. For this purpose, we utilized FACS technique to reveal fluorescence resonance energy transfer (FRET) in cells expressing wild type or LPI-mutant CFP-tagged y+LAT1 and YFP-tagged 4F2hc. The heteromerization of y+LAT1 and 4F2hc within the cell is not disrupted by any of the tested LPI mutations. In addition, the expression rate of the LPI mutant y+LAT1 proteins was significantly lower and cellular mortality was markedly increased than that of the wild type y+LAT1 in transfected samples. Our results indicate that the FACS-FRET method provides an alternative approach for screening of potential protein associations.

Heterodimerization of y(+)LAT-1 and 4F2hc visualized by acceptor photobleaching FRET microscopy.

y(+)LAT-1 and 4F2hc are the subunits of a transporter complex for cationic amino acids, located mainly in the basolateral plasma membrane of epithelial cells in the small intestine and renal tubules. Mutations in y(+)LAT-1 impair the transport function of this complex and cause a selective aminoaciduria, lysinuric protein intolerance (LPI, OMIM #222700), associated with severe, complex clinical symptoms. The subunits of an active transporter co-localize in the plasma membrane, but the exact process of dimerization is unclear since direct evidence for the assembly of this transporter in intact human cells has not been available. In this study, we used fluorescence resonance energy transfer (FRET) microscopy to investigate the interactions of y(+)LAT-1 and 4F2hc in HEK293 cells expressing y(+)LAT-1 and 4F2hc fused with ECFP or EYFP. FRET was quantified by measuring fluorescence intensity changes in the donor fluorophore (ECFP) after the photobleaching of the acceptor (EYFP). Increased donor fluorescence could be detected throughout the cell, from the endoplasmic reticulum and Golgi complex to the plasma membrane. Therefore, our data prove the interaction of y(+)LAT-1 and 4F2hc prior to the plasma membrane and thus provide evidence for 4F2hc functioning as a chaperone in assisting the transport of y(+)LAT-1 to the plasma membrane.

Promoter analysis of the human SLC7A7 gene encoding y+L amino acid transporter-1 (y+LAT-1).

The human SLC7A7 gene on chromosome 14q11.2 encodes the y+L amino acid transporter-1 (y+LAT-1) protein that transports, together with the 4F2hc cell surface antigen, cationic amino acids through the basolateral membrane of epithelial cells in the small intestine and kidney. The SLC7A7 gene comprises 11 exons, but the first two are not translated. Mutations in the coding region of the SLC7A7 gene cause a rare autosomal disorder, lysinuric protein intolerance (LPI). We have now investigated the expression levels and putative 5' promoter elements of the SLC7A7. The 5' region of the first untranslated exon contains no TATA-box, Inr elements nor other classical promoter elements, but has instead other putative transcription factor binding sequences. The E-box and AP-2 elements were able to bind proteins in HEK293 cells and adult kidney tissue extracts, but not in fibroblasts. Using transient transfection and luciferase reporter gene studies, we showed that the first two introns located in the untranslated region contained transcriptional enhancer elements. Northern blot analysis showed low and equal SLC7A7 mRNA levels in the control and LPI patient fibroblastoid and lymphoblast cells.

Expression of normal and mutant GFP-tagged y(+)L amino acid transporter-1 in mammalian cells.

Lysinuric protein intolerance (LPI; MIM 222700) is an autosomal recessive disorder characterized by defective transport of cationic amino acids lysine, arginine and ornithine. The defect is localized in the basolateral membrane of polar epithelial cells of the renal tubules and intestine. The SLC7A7 (solute carrier family 7, member 7) gene that encodes y(+)LAT-1 (y(+)L amino acid transporter-1) is mutated in LPI, and leads to the malfunction of the heterodimer composed of y(+)LAT-1 and 4F2hc (4F2 heavy chain) responsible for the system y(+)L amino acid transport activity at the membrane. In this study, the intracellular trafficking and membrane expression of wild type and four mutant y(+)LAT-1 proteins (LPI(Fin), G54V, 1548delC, W242X) was studied in two human cell lines by expressing green fluorescent protein (GFP) tagged proteins. Different SLC7A7 mutations influenced the trafficking of y(+)LAT-1 in the cells differently, as the wild type and missense mutant fusion proteins localized to the plasma membrane, while the frameshift and nonsense mutants sequestered to the cytoplasmic membranes, never reaching the target areas of the cell.