Whole exome sequencing of a novel homozygous missense variant in PALB2 gene leading to Fanconi anaemia complementation group.

Partner and localiser of BRCA2 (PALB2), also known as FANCN, is a key tumour suppressor gene in maintaining genome integrity. Monoallelic mutations of PALB2 are associated with breast and overian cancers, while bi-allelic mutations cause Fanconi anaemia (FA). In the present study, whole exome sequencing (WES) identified a novel homozygous missense variant, NM_024675.3: c.3296C>G (p.Thr1099Arg) in PALB2 gene (OMIM: 610355) that caused FA with mild pulmonary valve stenosis and dysmorphic and atypical features, including lymphangiectasia, non-immune hydrops fetalis and right-sided pleural effusion in a preterm female baby. WES results were further validated by Sanger sequencing. WES improves the screening and detection of novel and causative genetic variants to improve management of disease. To the best of our knowledge, the present study is the first reported FA case in a Saudi family with phenotypic atypical FA features. The results support the role of PALB2 gene and pathogenic variants that may cause clinical presentation of FA. Furthermore, the present results may establish a disease database, providing a groundwork for understanding the key genomic regions to control diseases resulting from consanguinity.

Combinatorial Therapeutic Potential of Stem Cells and Benzimidazol Derivatives for the Reduction of Liver Fibrosis.

(1) Background: Liver fibrosis is currently one of the top ten causes of death worldwide. Stem cells transplantation using mesenchymal stem cells (MSCs) is an alternative therapy which is used in the place of organ transplant, due to the incapacity of stem cells to endure oxidative stress in the damage site, thus affecting the healing process. The present study aimed to enhance the therapeutic potential of MSCs using combined therapy, along with the novel synthetic compounds of benzimidazol derivatives. (2) Methods: Eighteen compound series (benzimidazol derivatives) were screened against liver fibrosis using an in vitro CCl4-induced injury model on cultured hepatocytes. IC50 values were calculated on the bases of LDH assay and cell viability assay. (3) Results: Among the eighteen compounds, compounds (10), (14) and (18) were selected on the basis of IC50 value, and compound (10) was the most potent and had the lowest IC50 value in the LDH assay (8.399 ± 0.23 uM) and cell viability assay (4.73 ± 0.37 uM). Next, these compounds were combined with MSCs using an in vitro hepatocytes injury culture and in vivo rat fibrotic model. The effect of the MSCs + compounds treatment on injured hepatocytes was evaluated using LDH assay, cell viability assay, GSH assay and real-time PCR analysis and immuno-staining for caspase-3. Significant reductions in LDH level, caspase-3 and apoptotic marker genes were noted in MSCs + compounds-treated injured hepatocytes. In vivo data also showed the increased homing of the MSCs, along with compounds after transplantation. Real-time PCR analysis and TUNEL assay results also support our study. (4) Conclusions: It was concluded that compounds (10), (14) and (18) can be used in combination with MSCs to reduce liver fibrosis.

Two novel homozygous variants of ATP6V0A2 and ALDH18A1 lead to autosomal recessive cutis laxa type 2 and 3 in two Pakistani families.

BACKGROUND: Autosomal recessive cutis laxa type 2A (ARCL2A; OMIM: 219200) is characterized by neurovegetative, developmental and progeroid elastic skin anomalies. It is caused by biallelic variation in ATPase, H+ transporting V0 subunit A2 (ATP6V0A2; OMIM: 611716) located on chromosome 12q24.31. Autosomal recessive cutis laxa type 3A (ARCL3A; OMIM: 219150) is another subclinical type characterized by short stature, ophthalmological abnormalities and a progeria-like appearance. The ARCL3A is caused by loss of function alterations in the aldehyde dehydrogenase 18 family member A1 (ALDH18A1; OMIM: 138250) gene located at chromosome 10q24.1. METHODS: Whole-exome sequencing (WES), and Sanger sequencing were performed for molecular diagnosis. 3D protein modeling was performed to investigate the deleterious effect of the variant on protein structure. RESULTS: In this study, clinical and molecular diagnosis were performed for two families, ED-01 and DWF-41, which displayed hallmark features of ARCL2A and ARCL3A, respectively. Three affected individuals in the ED-01 family (IV-4, IV-5 and V-3) displayed sagging loose skin, down-slanting palpebral fissures, excessive wrinkles on the abdomen, hands and feet, and prominent veins on the trunk. Meanwhile the affected individuals in the DWF-41 family (V-2 and V-3) had progeroid skin, short stature, dysmorphology, low muscle tone, epilepsy, lordosis, scoliosis, delayed puberty and internal genitalia. WES in the index patient (ED-01: IV-4) identified a novel homozygous deletion (NM_012463.3: c.1977_1980del; p.[Val660LeufsTer23]) in exon 16 of the ATP6V0A2 while in DWF-41 a novel homozygous missense variant (NM_001323413.1:c.1867G>A; p.[Asp623Asn]) in exon 15 of the ALDH18A1 was identified. Sanger validation in all available family members confirmed the autosomal recessive modes of inheritances in each family. Three dimensional in-silico protein modeling suggested deleterious impact of the identified variants. Furthermore, these variants were assigned class 1 or "pathogenic" as per guidelines of American College of Medical Genetics 2015. Screening of ethnically matched healthy controls (n = 200 chromosomes), excluded the presence of these variations in general population. CONCLUSIONS: To the best of our knowledge, this is the first report of ATP6V0A2 and ALDH18A1 variations in the Pakhtun ethnicity of Pakistani population. The study confirms that WES can be used as a first-line diagnostic test in patients with cutis laxa, and provides basis for population screening and premarital testing to reduce the diseases burden in future generations.

A Novel Homozygous Nonsense Variant in the DYM Underlies Dyggve-Melchior-Clausen Syndrome in Large Consanguineous Family.

(1) Background: Dyggve-Melchior-Clausen Syndrome is a skeletal dysplasia caused by a defect in the DYM gene (OMIM number 607461). Pathogenic variants in the gene have been reported to cause Dyggve-Melchior-Clausen (DMC; OMIM 223800) dysplasia and Smith-McCort (SMC; OMIM 607326) dysplasia. (2) Methods: In the present study, large consanguineous families with five affected individuals with osteochondrodysplasia phenotypes were recruited. The family members were analyzed by polymerase chain reaction for homozygosity mapping using highly polymorphic microsatellite markers. Subsequent to linkage analysis, the coding exons and exon intron border of the DYM gene were amplified. The amplified products were then sent for Sanger sequencing. The structural effect of the pathogenic variant was analyzed by different bioinformatics tools. (3) Results: Homozygosity mapping revealed a 9 Mb homozygous region on chromosome 18q21.1 harboring DYM shared by all available affected individuals. Sanger sequencing of the coding exons and exon intron borders of the DYM gene revealed a novel homozygous nonsense variant [DYM (NM_017653.6):c.1205T>A, p.(Leu402Ter)] in affected individuals. All the available unaffected individuals were either heterozygous or wild type for the identified variant. The identified mutation results in loss of protein stability and weekend interactions with other proteins making them pathogenic (4) Conclusions: This is the second nonsense mutation reported in a Pakistani population causing DMC. The study presented would be helpful in prenatal screening, genetic counseling, and carrier testing of other members in the Pakistani community.