Novel TONSL variants cause SPONASTRIME dysplasia and associate with spontaneous chromosome breaks, defective cell proliferation and apoptosis.

SPONASTRIME dysplasia is an ultrarare spondyloepimetaphyseal dysplasia featuring short stature and short limbs, platyspondyly, depressed nasal bridge with midface hypoplasia and striated metaphyses. In 2019, an autosomal recessive inheritance was demonstrated by the identification of bi-allelic hypomorphic alleles in TONSL. The encoded protein has a critical role in maintaining genome integrity by promoting the homologous recombination required for repairing spontaneous replication-associated DNA lesions at collapsed replication forks. We report a 9-year-old girl with typical SPONASTRIME dysplasia and resulted in carrier of the novel missense p.(Gln430Arg) and p.(Leu1090Arg) variants in TONSL at whole-exome sequencing. In silico analysis predicted that these variants induced thermodynamic changes with a pathogenic impact on protein function. To support the pathogenicity of the identified variants, cytogenetic analysis and microscopy assays showed that patient-derived fibroblasts exhibited spontaneous chromosomal breaks and flow cytometry demonstrated defects in cell proliferation and enhanced apoptosis. These findings contribute to our understanding of the molecular pathogenesis of SPONASTRIME dysplasia and might open the way to novel therapeutic approaches.

A single-center study on 140 patients with cerebral cavernous malformations: 28 new pathogenic variants and functional characterization of a PDCD10 large deletion.

Cerebral cavernous malformation (CCM) is a capillary malformation arising in the central nervous system. CCM may occur sporadically or cluster in families with autosomal dominant transmission, incomplete penetrance, and variable expressivity. Three genes are associated with CCM KRIT1, CCM2, and PDCD10. This work is a retrospective single-center molecular study on samples from multiple Italian clinical providers. From a pool of 317 CCM index patients, we found germline variants in either of the three genes in 80 (25.2%) probands, for a total of 55 different variants. In available families, extended molecular analysis found segregation in 60 additional subjects, for a total of 140 mutated individuals. From the 55 variants, 39 occurred in KRIT1 (20 novel), 8 in CCM2 (4 novel), and 8 in PDCD10 (4 novel). Effects of the three novel KRIT1 missense variants were characterized in silico. We also investigated a novel PDCD10 deletion spanning exon 4-10, on patient's fibroblasts, which showed significant reduction of interactions between KRIT1 and CCM2 encoded proteins and impaired autophagy process. This is the largest study in Italian CCM patients and expands the known mutational spectrum of KRIT1, CCM2, and PDCD10. Our approach highlights the relevance of seeking supporting information to pathogenicity of new variants for the improvement of management of CCM.

Pontocerebellar hypoplasia type 6 caused by mutations in RARS2: definition of the clinical spectrum and molecular findings in five patients.

Recessive mutations in the mitochondrial arginyl-transfer RNA synthetase (RARS2) gene have been associated with early onset encephalopathy with signs of oxidative phosphorylation defects classified as pontocerebellar hypoplasia 6. We describe clinical, neuroimaging and molecular features on five patients from three unrelated families who displayed mutations in RARS2. All patients rapidly developed a neonatal or early-infantile epileptic encephalopathy with intractable seizures. The long-term follow-up revealed a virtual absence of psychomotor development, progressive microcephaly, and feeding difficulties. Mitochondrial respiratory chain enzymes in muscle and fibroblasts were normal in two. Blood and CSF lactate was abnormally elevated in all five patients at early stages while appearing only occasionally abnormal with the progression of the disease. Cerebellar vermis hypoplasia with normal aspect of the cerebral and cerebellar hemispheres appeared within the first months of life at brain MRI. In three patients follow-up neuroimaging revealed a progressive pontocerebellar and cerebral cortical atrophy. Molecular investigations of RARS2 disclosed the c.25A>G/p.I9V and the c.1586+3A>T in family A, the c.734G>A/p.R245Q and the c.1406G>A/p.R469H in family B, and the c.721T>A/p.W241R and c.35A>G/p.Q12R in family C. Functional complementation studies in Saccharomyces cerevisiae showed that mutation MSR1-R531H (equivalent to human p.R469H) abolished respiration whereas the MSR1-R306Q strain (corresponding to p.R245Q) displayed a reduced growth on non-fermentable YPG medium. Although mutations functionally disrupted yeast we found a relatively well preserved arginine aminoacylation of mitochondrial tRNA. Clinical and neuroimaging findings are important clues to raise suspicion and to reach diagnostic accuracy for RARS2 mutations considering that biochemical abnormalities may be absent in muscle biopsy.

20 novel point mutations and one large deletion in EXT1 and EXT2 genes: report of diagnostic screening in a large Italian cohort of patients affected by hereditary multiple exostosis.

BACKGROUND: Hereditary multiple exostosis represents the most frequent bone tumor disease in humans. It consists of cartilage deformities affecting the juxta-ephyseal region of long bones. Usually benign, exostosis could degenerate in malignant chondrosarcoma form in less than 5% of the cases. Being caused by mutations in the predicted tumor suppressor genes, EXT1 (chr 8q23-q24) and EXT2 (chr 11p11-p12) genes, HMEs are usually inherited with an autosomal dominant pattern, although "de novo" cases are not infrequent. AIM: Here we present our genetic diagnostic report on the largest Southern Italy cohort of HME patients consisting of 90 subjects recruited over the last 5years. RESULTS: Molecular screening performed by direct sequencing of both EXT1 and EXT2 genes, by MLPA and Array CGH analyses led to the identification of 66 mutations (56 different occurrences) and one large EXT2 deletion out of 90 patients (74.4%). The total of 21 mutations (20 different occurrences, 33.3%) and the EXT2 gene deletion were novel. In agreement with literature data, EXT1 gene mutations were scattered along all the protein sequence, while EXT2 lesions fell in the first part of the protein. Conservation, damaging prediction and 3-D modeling, in-silico, analyses, performed on three novel missense variants, confirmed that at least in two cases the novel aminoacidic changes could alter the structure stability causing a strong protein misfolding. CONCLUSIONS: Here we present 20 novel EXT1/EXT2 mutations and one large EXT2 deletion identified in the largest Southern Italy cohort of patients affected by hereditary multiple exostosis.

Mutational screening of VSX1, SPARC, SOD1, LOX, and TIMP3 in keratoconus.

PURPOSE: To evaluate the involvement of Visual System Homeobox 1 (VSX1), Secreted Protein Acidic and Rich in Cysteine (SPARC), Superoxide Dismutase 1 (SOD1), Lysyl Oxidase (LOX), and Tissue Inhibitor of Metalloproteinase 3 (TIMP3) in sporadic and familial keratoconus. METHODS: Mutational analysis of the five genes was performed by sequencing and fragment analysis in a large cohort of 302 Italian patients, with a diagnosis of keratoconus based on clinical examination and corneal topography. The variants identified in VSX1 and SPARC were also assessed in the available relatives of the probands. RESULTS: A novel mutation p.G239R and previously reported mutations were found in VSX1. Novel and already reported variants were identified in SPARC and SOD1, whose pathogenic significance has not been established. No pathogenic variants have been identified in LOX and TIMP3. CONCLUSIONS: Molecular analysis of the five genes in a cohort of 225 sporadic and 77 familial keratoconus cases confirms the possible pathogenic role of VSX1 though in a small number of patients; a possible involvement of LOX and TIMP3 could be excluded; and the role played by SOD1 and SPARC in determining the disease as not been definitively clarified. Further studies are required to identify other important genetic factors involved in the pathogenesis and progression of the disease that in the authors' opinion, and according with several authors, should be considered as a complex disease.

Large rearrangements detected by MLPA, point mutations, and survey of the frequency of mutations within the SLC3A1 and SLC7A9 genes in a cohort of 172 cystinuric Italian patients.

Cystinuria is a rare inherited disorder characterized by defective renal reabsorption of cystine and the dibasic amino acids. SLC3A1 and SLC7A9 have been identified as responsible genes. The large majority of the more than 200 mutations so far identified in the two genes are point mutations, while only few alleles carrying gross genomic alterations have been reported. We screened 39 cystinuric patients for large rearrangements, by two home-made multiplex ligation-dependent probe amplification (MLPA) assays. MLPA analysis led to the identification of 6 different alleles in SLC3A1 and 2 in SLC7A9 accounting for a total of 25 copy number changes, 11 in SLC3A1 and 14 in SLC7A9. Three large rearrangements in SLC3A1, deletion of exons 2-4 (E2_E4del), deletion of exons 5-6 (E5_E6del) and duplication of exons 8-9 (E8_E9dup) are novel. A complete SLC7A9 gene deletion was found in three patients. In addition, we report the identification of three novel point mutations in SLC7A9 (p.G105E, p.R250K, c.1416_1417insAC), the frequency and the occurrence of cystinuria mutations in a cohort of 172 Italian patients. In conclusion, we developed a reliable and robust MLPA analytic method for SLC3A1 and SLC7A9 genes that represents an optimal complement to DNA sequence analysis in patients with cystinuria, enabling the screening for deletions and duplications.

Twenty-four novel mutations identified in a cohort of 85 patients by direct sequencing of the SLC3A1 and SLC7A9 cystinuria genes.

Mutations in the SLC3A1 and SLC7A9 genes cause cystinuria (OMIM 220100), an autosomal recessive disorder of amino acid transport and reabsorption in the proximal renal tubule and in the epithelial cells of the gastrointestinal tract. In an attempt to characterize the molecular defect in the SLC3A1 and SLC7A9 genes, we analyzed a cohort of 85 unrelated subjects clinically diagnosed as affected by cystinuria on the basis of stone formation, prevalently of Italian and Greek origin. Analysis of all coding region and exon-intron junctions of the SLC3A1 and SLC7A9 genes by using direct sequencing method allowed us to identify 62 different mutations in 83 out of 85 patients accounting for 90.5% of all affected chromosomes. Twenty-four out of 62 are novel mutations, 9 in SLC3A1 and 15 in SLC7A9. In conclusion, this report expands the spectrum of SLC3A1 and SLC7A9 mutations and confirms the heterogeneity of this disorder.

A combined approach to the molecular analysis of cystinuria: from urinalysis to sequencing via genotyping.

BACKGROUND: Cystinuria is an autosomal recessive disease that is manifested by kidney stones and is caused by mutations in two genes: SLC3AI on chromosome 2p and SLC7A9 on chromosome 19q. Urinary cystine levels in obligate carriers are often, but not always, helpful in identifying the causative gene. OBJECTIVES: To characterize the clinical features and analyze the genetic basis of cystinuria in an inbred Moslem Arab Israeli family. METHODS: Family members were evaluated for urinary cystine and amino acid levels. DNA was initially analyzed with polymorphic markers close to the two genes and SLC7A9 was fully sequenced. RESULTS: Full segregation was found with the marker close to SLC7A9. Sequencing of this gene revealed a missense mutation, P482L, in the homozygous state in all three affected sibs. CONCLUSIONS: A combination of urinary cystine levels in obligate carriers, segregation analysis with polymorphic markers, and sequencing can save time and resources in the search for cystinuria mutations.