Monoallelic pathogenic ALG5 variants cause atypical polycystic kidney disease and interstitial fibrosis.

Disorders of the autosomal dominant polycystic kidney disease (ADPKD) spectrum are characterized by the development of kidney cysts and progressive kidney function decline. PKD1 and PKD2, encoding polycystin (PC)1 and 2, are the two major genes associated with ADPKD; other genes include IFT140, GANAB, DNAJB11, and ALG9. Genetic testing remains inconclusive in ∼7% of the families. We performed whole-exome sequencing in a large multiplex genetically unresolved (GUR) family affected by ADPKD-like symptoms and identified a monoallelic frameshift variant (c.703_704delCA) in ALG5. ALG5 encodes an endoplasmic-reticulum-resident enzyme required for addition of glucose molecules to the assembling N-glycan precursors. To identify additional families, we screened a cohort of 1,213 families with ADPKD-like and/or autosomal-dominant tubulointerstitial kidney diseases (ADTKD), GUR (n = 137) or naive to genetic testing (n = 1,076), by targeted massively parallel sequencing, and we accessed Genomics England 100,000 Genomes Project data. Four additional families with pathogenic variants in ALG5 were identified. Clinical presentation was consistent in the 23 affected members, with non-enlarged cystic kidneys and few or no liver cysts; 8 subjects reached end-stage kidney disease from 62 to 91 years of age. We demonstrate that ALG5 haploinsufficiency is sufficient to alter the synthesis of the N-glycan chain in renal epithelial cells. We also show that ALG5 is required for PC1 maturation and membrane and ciliary localization and that heterozygous loss of ALG5 affects PC1 maturation. Overall, our results indicate that monoallelic variants of ALG5 lead to a disorder of the ADPKD-spectrum characterized by multiple small kidney cysts, progressive interstitial fibrosis, and kidney function decline.

Splicing analysis of SLC40A1 missense variations and contribution to hemochromatosis type 4 phenotypes.

Hemochromatosis type 4, or ferroportin disease, is considered as the second leading cause of primary iron overload after HFE-related hemochromatosis. The disease, which is predominantly associated with missense variations in the SLC40A1 gene, is characterized by wide clinical heterogeneity. We tested the possibility that some of the reported missense mutations, despite their positions within exons, cause splicing defects. Fifty-eight genetic variants were selected from the literature based on two criteria: a precise description of the nucleotide change and individual evidence of iron overload. The selected variants were investigated by different in silico prediction tools and prioritized for midigene splicing assays. Of the 15 variations tested in vitro, only two were associated with splicing changes. We confirm that the c.1402G>A transition (p.Gly468Ser) disrupts the exon 7 donor site, leading to the use of an exonic cryptic splicing site and the generation of a truncated reading frame. We observed, for the first time, that the p.Gly468Ser substitution has no effect on the ferroportin iron export function. We demonstrate alternative splicing of exon 5 in different cell lines and show that the c.430A>G (p.Asn144Asp) variant promotes exon 5 inclusion. This could be part of a gain-of-function mechanism. We conclude that splicing mutations rarely contribute to hemochromatosis type 4 phenotypes. An in-depth investigation of exon 5 auxiliary splicing sequences may help to elucidate the mechanism by which splicing regulatory proteins regulate the production of the full length SLC40A1 transcript and to clarify its physiological importance.

Nation-wide investigation of RHD variants in Thai blood donors: Impact for molecular diagnostics.

BACKGROUND: Knowledge of the molecular determinants driving antigen expression is critical to design, optimize, and implement a genotyping approach on a population-specific basis. Although RHD gene variability has been extensively reported in Caucasians, Africans, and East-Asians, it remains to be explored in Southeast Asia. Thus the molecular basis of non-D+ blood donors was investigated in Thailand. STUDY DESIGN AND METHODS: First, 1176 blood samples exhibiting an inconclusive or negative result by automated serological testing were collected in the 12 Regional Blood Centres of the Thai Red Cross located throughout Thailand. Second, the RHD gene was analyzed in all samples by 1) quantitative multiplex PCR of short fluorescent fragments, and 2) direct sequencing, when necessary, for identifying structural variants and single nucleotide variants, respectively. RESULTS: Additional serological typing yielded 51 and 1125 samples with weak/partial D and D-negative (D-) phenotype, respectively. In the first subset, partial RHD*06.03 was the most common variant allele (allele frequency: 18.6%). In the second subset, the whole deletion of the gene is largely the most frequent (allele frequency: 84.9%), followed by the Asian DEL allele found in 15.6% of the samples. Eight novel alleles with various mutational mechanisms were identified. CONCLUSION: We report, for the first time at the national level, the molecular basis of weak/partial D and serologically D- phenotypes in Thai blood donors. The design and implementation of a dedicated diagnostic strategy in blood donors and patients are the very next steps for optimizing the management and supply of RBC units in Thailand.

Missense RHD single nucleotide variants induce weakened D antigen expression by altering splicing and/or protein expression.

BACKGROUND: Although D variant phenotype is known to be due to genetic defects, including rare missense single nucleotide variants (SNVs), within the RHD gene, few studies have addressed the molecular and cellular mechanisms driving this altered expression. We and others showed previously that splicing is commonly disrupted by SNVs in constitutive splice sites and their vicinity. We thus sought to investigate whether rare missense SNVs located in "deep" exonic regions could also impair this mechanism. STUDY DESIGN AND METHODS: Forty-six missense SNVs reported within exons 6 and 7 were first selected from the Human RhesusBase. Their respective effect on splicing was assessed by using an in vitro assay. An RhD-negative cell model was further generated by using the CRISPR-Cas9 approach. RhD-mutated proteins were overexpressed in the newly created model, and cell membrane expression of the D antigen was measured by flow cytometry. RESULTS: Minigene splicing assay showed that 14 of 46 (30.4%) missense SNVs alter splicing. Very interestingly, further investigation of two missense SNVs, which both affect codon 338 and confer a weak D phenotype, showed various mechanisms: c.1012C>G (p.Leu338Val) disrupts splicing only, while c.1013T>C (p.Leu338Pro) alters only the protein structure, in agreement with in silico prediction tools and 3D protein structure visualization. CONCLUSION: Our functional data set suggests that missense SNVs damage quantitatively D antigen expression by, at least, two different mechanisms (splicing alteration and protein destabilization) that may act independently. These data thereby contribute to extend the current knowledge of the molecular mechanisms governing weakened D expression.

Comprehensive Molecular Analysis of Serologically D-Negative and Weak/Partial D Phenotype in Thai Blood Donors.

BACKGROUND: Molecular genetics of the Rh system has been extensively studied in Caucasians, Black Africans, East Asians, and Indians more recently. In this work, we sought to investigate the molecular basis of variant D expression in the Thai population, which remains unknown. MATERIALS AND METHODS: Blood samples from 450 Thai donors showing the variant D phenotype were collected. The RHD gene was analyzed by quantitative multiplex polymerase chain reaction of short fluorescent fragments and/or Sanger sequencing. RESULTS: The most frequent alleles in 200 D-negative and 121 DEL samples were the whole RHD gene deletion and the Asian DEL alleles, respectively. In 129 weak/partial D samples, 36 variant alleles were identified, including eight novel alleles. RHD*06.03, which is common in variant D samples from South China, is the most prevalent variant allele, followed by the recently reported Indian RHD*01W.150 allele. DISCUSSION: For the first time, a comprehensive overview of the nature and distribution of variant RHD alleles in Thailand is reported. It is a milestone to pave the way towards improvement of the current screening strategy to identify DEL donors accurately. The next step will be the design and implementation of a simple molecular test for screening the most frequent alleles, specifically in this population.

First estimate of the scale of canonical 5' splice site GT>GC variants capable of generating wild-type transcripts.

It has long been known that canonical 5' splice site (5'SS) GT>GC variants may be compatible with normal splicing. However, to date, the actual scale of canonical 5'SSs capable of generating wild-type transcripts in the case of GT>GC substitutions remains unknown. Herein, combining data derived from a meta-analysis of 45 human disease-causing 5'SS GT>GC variants and a cell culture-based full-length gene splicing assay of 103 5'SS GT>GC substitutions, we estimate that ~15-18% of canonical GT 5'SSs retain their capacity to generate between 1% and 84% normal transcripts when GT is substituted by GC. We further demonstrate that the canonical 5'SSs in which substitution of GT by GC-generated normal transcripts exhibit stronger complementarity to the 5' end of U1 snRNA than those sites whose substitutions of GT by GC did not lead to the generation of normal transcripts. We also observed a correlation between the generation of wild-type transcripts and a milder than expected clinical phenotype but found that none of the available splicing prediction tools were capable of reliably distinguishing 5'SS GT>GC variants that generated wild-type transcripts from those that did not. Our findings imply that 5'SS GT>GC variants in human disease genes may not invariably be pathogenic.

Functional analysis of novel RHD variants: splicing disruption is likely to be a common mechanism of variant D phenotype.

BACKGROUND: We previously showed that several variations in the RHD gene, including synonymous changes, can be classified as splice site variants and may play a direct role in D variant phenotype expression. We sought to extend our study to additional candidates, notably in the first and last exons of the gene, by engineering a novel universal splice reporting vector, i.e., minigene. STUDY DESIGN AND METHODS: Our previous plasmid construct was modified to allow subcloning of any exon(s) of interest for assessing effect of variations on splicing. Seventeen novel and/or uncharacterized variations of the RHD gene were selected for the study and tested in our novel model. RESULTS: We engineered and validated a novel universal minigene for assessing virtually any variations of interest for splicing defect. Of the 17 variants tested in the novel model, 11 were shown to alter splicing either totally or partially, including the silent c.1065C>T variation, which induces major skipping of exon 7, and may therefore be responsible for reducing D antigen expression. We also showed that while all three missense variations c.1154G>C, c.1154G>T, and c.1154G>A in exon 9 are splice site variants, splicing is differentially altered and D-negative phenotype observed in the presence of the latter substitution is likely due to a defect in RhD protein folding. CONCLUSION: Overall, we hypothesize that splicing alteration is likely to be a common mechanism of D phenotype variation that has been underestimated so far. Further large-scale studies are necessary to demonstrate this statement definitely.