Improving mutation screening in familial hematuric nephropathies through next generation sequencing.
Alport syndrome is an inherited nephropathy associated with mutations in genes encoding type IV collagen chains present in the glomerular basement membrane. COL4A5 mutations are associated with the major X-linked form of the disease, and COL4A3 and COL4A4 mutations are associated with autosomal recessive and dominant forms (thought to be involved in 15% and 1%-5% of the families, respectively) and benign familial hematuria. Mutation screening of these three large genes is time-consuming and expensive. Here, we carried out a combination of multiplex PCR, amplicon quantification, and next generation sequencing (NGS) analysis of three genes in 101 unrelated patients. We identified 88 mutations and 6 variations of unknown significance on 116 alleles in 83 patients. Two additional indel mutations were found only by secondary Sanger sequencing, but they were easily identified retrospectively with the web-based sequence visualization tool Integrative Genomics Viewer. Altogether, 75 mutations were novel. Sequencing the three genes simultaneously was particularly advantageous as the mode of inheritance could not be determined with certainty in many instances. The proportion of mutations in COL4A3 and COL4A4 was notably high, and the autosomal dominant forms of Alport syndrome appear more frequently than reported previously. Finally, this approach allowed the identification of large COL4A3 and COL4A4 rearrangements not described previously. We conclude that NGS is efficient, reduces screening time and cost, and facilitates the provision of appropriate genetic counseling in Alport syndrome.
Departments of Genetics, and Assistance Publique des Hôpitaux de Paris, Reference Center for Renal Hereditary Disease for Children and Adults (MARHEA), Paris, France;
Vincent
V
Morinière
Department of Genetics, Institute of Pathology and Genetics, Gosselies, Belgium;
Karin
K
Dahan
Department of Genetics, Institute of Pathology and Genetics, Gosselies, Belgium;
Pascale
P
Hilbert
Department of Genetics, Institute of Pathology and Genetics, Gosselies, Belgium;
Marieline
M
Lison
Assistance Publique des Hôpitaux de Paris, Reference Center for Renal Hereditary Disease for Children and Adults (MARHEA), Paris, France;
Said
S
Lebbah
Department of Clinical Genetics, Sahlgrenska University Hospital, Gothenburg, Sweden;
Alexandra
A
Topa
Genomics Platform, Imagine Institute, Paris, France;
Christine
C
Bole-Feysot
Genomics Platform, Imagine Institute, Paris, France;
Solenn
S
Pruvost
Bioinformatics Platform, Paris Descartes-Sorbonne Paris Cité University, Paris, France;
Patrick
P
Nitschke
Assistance Publique des Hôpitaux de Paris, Nephrology Service, Tenon Hospital, Paris, France;
Emmanuelle
E
Plaisier
Nephrology Service and.
Bertrand
B
Knebelmann
Assistance Publique des Hôpitaux de Paris, Pediatric Nephrology Service, Robert Debré Hospital, Paris, France; and.
Marie-Alice
MA
Macher
Pathology, and.
Laure-Hélène
LH
Noel
Institut National de la Santé et de la Recherche Médicale, Inserm UMR 1163, Laboratory of Inherited Kidney Diseases, Imagine Institute, Paris, France.
Marie-Claire
MC
Gubler
Departments of Genetics, and Assistance Publique des Hôpitaux de Paris, Reference Center for Renal Hereditary Disease for Children and Adults (MARHEA), Paris, France; Institut National de la Santé et de la Recherche Médicale, Inserm UMR 1163, Laboratory of Inherited Kidney Diseases, Imagine Institute, Paris, France Paris Descartes-Sorbonne Paris Cité University, and corinne.antignac@inserm.fr.
Corinne
C
Antignac
Assistance Publique des Hôpitaux de Paris, Reference Center for Renal Hereditary Disease for Children and Adults (MARHEA), Paris, France; Pediatric Nephrology Service, Assistance Publique des Hôpitaux de Paris, Necker-Enfants Malades Hospital, Paris, France;
Laurence
L
Heidet
D000293:Adolescent; D000328:Adult; D001324:Autoantigens / Q000235:genetics; D002648:Child; D002675:Child, Preschool; D015331:Cohort Studies; D024141:Collagen Type IV / Q000235:genetics; D004252:DNA Mutational Analysis; D005192:Family Health; D005260:Female; D005817:Genetic Counseling; D006579:Heterozygote; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D009394:Nephritis, Hereditary / Q000235:genetics* / Q000473:pathology*; D010641:Phenotype; D020641:Polymorphism, Single Nucleotide; D055815:Young Adult
D001324:Autoantigens; C466620:COL4A4 protein, human; C478159:COL4A5 protein, human; D024141:Collagen Type IV; C057112:type IV collagen alpha3 chain
Alport syndrome; genetic renal disease; molecular genetics