Next generation sequencing as a useful tool in the diagnostics of mosaicism in Alport syndrome

Beicht, Sonja; Strobl‐Wildemann, Gertrud; Rath, Sabine; Wachter, Oliver; Alberer, Martin; Kaminsky, Elke; Weber, Lutz T.; Hinrichsen, Tanja; Klein, Hanns‐Georg; Hoefele, Julia

doi:10.1016/j.gene.2013.05.045

Cited by 34 publications

(23 citation statements)

References 21 publications

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“…A recent case study reported a high sensitivity for detecting somatic mosaic mutations in the COL4A5 gene using nextgeneration sequencing (24). High-throughput sequencing technologies may not only lower the cost of DNA ID 128.…”

Section: Discussionmentioning

confidence: 99%

X-Linked Alport Syndrome Caused by Splicing Mutations in COL4A5

Nozu

Vořechovský

Kono

et al. 2014

Clinical Journal of the American Society of Nephrology

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Background and objectives X-linked Alport syndrome is caused by mutations in the COL4A5 gene. Although many COL4A5 mutations have been detected, the mutation detection rate has been unsatisfactory. Some men with X-linked Alport syndrome show a relatively mild phenotype, but molecular basis investigations have rarely been conducted to clarify the underlying mechanism.Design, setting, participants, & measurements In total, 152 patients with X-linked Alport syndrome who were suspected of having Alport syndrome through clinical and pathologic investigations and referred to the hospital for mutational analysis between January of 2006 and January of 2013 were genetically diagnosed. Among those patients, 22 patients had suspected splice site mutations. Transcripts are routinely examined when suspected splice site mutations for abnormal transcripts are detected; 11 of them showed expected exon skipping, but others showed aberrant splicing patterns. The mutation detection strategy had two steps: (1) genomic DNA analysis using PCR and direct sequencing and (2) mRNA analysis using RT-PCR to detect RNA processing abnormalities.Results Six splicing consensus site mutations resulting in aberrant splicing patterns, one exonic mutation leading to exon skipping, and four deep intronic mutations producing cryptic splice site activation were identified. Interestingly, one case produced a cryptic splice site with a single nucleotide substitution in the deep intron that led to intronic exonization containing a stop codon; however, the patient showed a clearly milder phenotype for X-linked Alport syndrome in men with a truncating mutation. mRNA extracted from the kidney showed both normal and abnormal transcripts, with the normal transcript resulting in the milder phenotype. This novel mechanism leads to mild clinical characteristics.Conclusions This report highlights the importance of analyzing transcripts to enhance the mutation detection rate and provides insight into genotype-phenotype correlations. This approach can clarify the cause of atypically mild phenotypes in X-linked Alport syndrome.

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Section: Discussionmentioning

confidence: 99%

X-Linked Alport Syndrome Caused by Splicing Mutations in COL4A5

Nozu

Vořechovský

Kono

et al. 2014

Clinical Journal of the American Society of Nephrology

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“…Mosaicism has been previously reported in patients with mild presentation of mendelian disorders. 18 We therefore suspected that Sanger sequencing might have missed the mutation in the father because of low-grade somatic mosaicism.…”

Section: Clinical Presentations Of Cases With Pathogenic Adcy5mentioning

confidence: 99%

ADCY5 mutations are another cause of benign hereditary chorea

et al. 2015

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Objective: To determine the contribution of ADCY5 mutations in cases with genetically undefined benign hereditary chorea (BHC). Methods:We studied 18 unrelated cases with BHC (7 familial, 11 sporadic) who were negative for NKX2-1 mutations. The diagnosis of BHC was based on the presence of a childhood-onset movement disorder, predominantly characterized by chorea and no other major neurologic features. ADCY5 analysis was performed by whole-exome sequencing or Sanger sequencing. ADCY5 and NKX2-1 expression during brain development and in the adult human brain was assessed using microarray analysis of postmortem brain tissue.Results: The c.1252C.T; p.R418W mutation was identified in 2 cases (1 familial, 1 sporadic).The familial case inherited the mutation from the affected father, who had a much milder presentation, likely due to low-grade somatic mosaicism. The mutation was de novo in the sporadic case. The clinical presentation of these cases featured nonparoxysmal generalized chorea, as well as dystonia in the most severely affected, but no facial myokymia. We observed significant progression of symptoms in ADCY5 mutation carriers, in contrast to BHC secondary to NKX2-1 mutations. The difference in the clinical course is mirrored by the brain expression data, showing increasing ADCY5 expression in the striatum during brain development, whereas NKX2-1 shows an opposite trend.Conclusions: Our study identifies mutations in ADCY5, the gene previously linked to familial dyskinesia with facial myokymia, as a cause of familial and sporadic BHC. ADCY5 genetic analysis should be performed in cases with a benign choreiform movement disorder even in the absence of facial myokymia. Benign hereditary chorea (BHC) (OMIM 118700) is a rare and poorly delineated syndrome, clinically characterized by onset of symptoms in infancy or early childhood, relatively little clinical progression, and absence of other major neurologic deficits, in particular prominent cognitive decline.

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“…7 To date, however, only six patients in four reports have been described with somatic mosaic variants in COL4A5, including our previous report (Table 4). 7,[19][20][21] Although all six cases showed a milder phenotype and some of the female cases were asymptomatic, no asymptomatic male cases have previously been reported. A recent publication by Beicht et al 19 described an asymptomatic female XLAS patient with a somatic mosaic variant who had variant allele frequencies of 14, 7, 4, and 7% in leukocytes, urine sediments, hair roots, and oral mucosa, respectively, as shown by NGS.…”

Section: Discussionmentioning

confidence: 89%

“…7,[19][20][21] Although all six cases showed a milder phenotype and some of the female cases were asymptomatic, no asymptomatic male cases have previously been reported. A recent publication by Beicht et al 19 described an asymptomatic female XLAS patient with a somatic mosaic variant who had variant allele frequencies of 14, 7, 4, and 7% in leukocytes, urine sediments, hair roots, and oral mucosa, respectively, as shown by NGS. However, it is difficult to evaluate variant allele frequencies and phenotypes in female XLAS patients because skewed X-inactivation might affect the phenotype.…”

Section: Discussionmentioning

confidence: 89%

Somatic mosaicism and variant frequency detected by next-generation sequencing in X-linked Alport syndrome

Nozu

Kono

et al. 2015

Eur J Hum Genet

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X-linked Alport syndrome (XLAS) is a progressive, hereditary nephropathy. Although men with XLAS usually develop end-stage renal disease before 30 years of age, some men show a milder phenotype and develop end-stage renal disease later in life. However, the molecular mechanisms associated with this milder phenotype have not been fully identified. We genetically diagnosed 186 patients with suspected XLAS between January 2006 and August 2014. Genetic examination involved: (1) extraction and analysis of genomic DNA using PCR and direct sequencing using Sanger's method and (2) next-generation sequencing to detect variant allele frequencies. We identified somatic mosaic variants in the type VI collagen, α5 gene (COL4A5) in four patients. Interestingly, two of these four patients with variant frequencies in kidney biopsies or urinary sediment cells of ≥ 50% showed hematuria and moderate proteinuria, whereas the other two with variant frequencies of o50% were asymptomatic or only had hematuria. De novo variants can occur even in asymptomatic male cases of XLAS resulting in mosaicism, with important implications for genetic counseling. This is the first study to show a tendency between the variant allele frequency and disease severity in male XLAS patients with somatic mosaic variants in COL4A5. Although this is a very rare status of somatic mosaicism, further analysis is needed to show this correlation in a larger population.

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Next generation sequencing as a useful tool in the diagnostics of mosaicism in Alport syndrome

Cited by 34 publications

References 21 publications

X-Linked Alport Syndrome Caused by Splicing Mutations in COL4A5

X-Linked Alport Syndrome Caused by Splicing Mutations in COL4A5

ADCY5 mutations are another cause of benign hereditary chorea

Somatic mosaicism and variant frequency detected by next-generation sequencing in X-linked Alport syndrome

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