Identification and functional characterization of a novel truncating splicing variant in COL4A5 gene causes X-linked Alport syndrome with astigmatism

To the Editor: Alport syndrome (AS) is a rare hereditary progressive nephropathy manifested with hematuria, proteinuria, gradual renal failure leads to end-stage renal disease (ESRD), sensorineural hearing loss, and ocular abnormalities.^[1] Germline mutations in any of these collagen type IV genes (COL4A3, COL4A4, and COL4A5) cause AS.^[1] Here, we investigated a four-generation Chinese family with AS. The proband was clinically diagnosed with hematuria and proteinuria with an elevated level of plasma creatinine. Later, the proband was diagnosed with ESRD and received renal transplantation. Unlike other AS patients, the proband was identified with astigmatism. Whole exome sequencing identified a hemizygous novel splicing variant (c.4688 + 2T > C) in intron 48 of COL4A5 gene in the proband. This variant causes aberrant splicing of COL4A5 mRNA, followed by the skipping of exon 48, leads to frameshift (p.G1504Dfs∗11) which predicted to form a truncated COL4A5 protein. Our study reported a truncating splicing variant in COL4A5 causes X-linked AS (XLAS) in a four-generation family. The study was approved by the Ethics Committee of Bao’an Women and Children's Hospital (No. LLSC2016-06002) in compliance with the Helsinki Declaration of Principles. Informed consent was obtained from all the family members.

Here, a four-generation Chinese family with AS was recruited and investigated. The proband (III:6), a 24-year-old man was clinically diagnosed with hematuria and proteinuria [Figure 1A]. He was clinically diagnosed with hematuria and proteinuria in the first year of his life. At the age of 18 years, he was identified with an elevated level of plasma creatinine and gradually and progressively developed to ESRD (Cr: 751.8 μmol/L). Three years later, he received renal transplantation. In addition, he showed no other ophthalmic symptoms except astigmatism. Proband's elder brother (III:5) died from ESRD when he was 14 years old. The proband's mother (II:8) was diagnosed with proteinuria at the age of 40 years. Her plasma creatinine level started to increase when she was 48 years old and one year later, she was identified with hearing impairment of the right ear. Later, she has been identified with ESRD (Cr: 800.0 μmol/L) and received renal transplantation at the age of 50 years. Proband's cousin sister (III:1) also received renal transplantation at the age of 32 years and the renal function of her mother (II:2) was found normal. Proband's matrilineal grandmother (I:2) and aunt (II:4) were not identified with ESRD and died due to an accident.

Figure 1: (A) Pedigrees of a four-generation family with AS. Squares and circles denoted males and females, respectively. Black symbols indicate affected individuals; white symbols indicate unaffected family members. Arrow indicates the proband (III:6). Slashed symbols indicate deceased subjects. (B) Partial DNA sequences in the COL4A5 gene by Sanger sequencing of the family. The reference sequence NM_000495 of the COL4A5 gene was used. (C) RT-PCR products of the c.4688 + 2T > C in COL4A5, Lane Marker: the 1000 bp marker/ladder. Lane 1: the WT correctly spliced exon 47, exon 48, and exon 49 of COL4A5 cDNA in proband's father. Lane 2: proband's mother with the WT correctly spliced exon 47, exon 48, and exon 49 of COL4A5 cDNA as well as identified with the aberrantly spliced COL4A5 cDNA with complete skipping of exon 48. Lane 3: the aberrantly spliced COL4A5 cDNA with complete skipping of exon 48. (D) RT-PCR and direct sequencing of COL4A5 cDNA showed the unaffected or normal family member with normal splicing of exon 47 to exon 49. In contrast, Sanger sequencing of mutant RT-PCR products revealed complete loss of exon 48 due to abolition of the WT donor splice site. (E) Schematic representation of the splicing event in both the proband as well as the unaffected family members. AS: Alport syndrome; cDNART-PCR: Reverse transcription polymerase chain reaction; WT: Wild-type.

Proband's genomic DNA was subjected to whole exome sequencing. Whole exome sequencing identified a hemizygous novel splicing variant (c.4688 + 2T > C) in intron 48 of the COL4A5 gene in the proband [Figure 1B]. Sanger sequencing confirmed that this splicing variant was co-segregated well among all the affected family members as well as absent in all the unaffected family members and 200 ethnically matched normal individuals. This variant is also absent in the Human Gene Variant database (www.hgmd.cf.ac.uk/), Online Mendelian Inheritance in Man (https://www.omim.org), our in-house database (consisting of ∼50,000 Chinese samples), ExAC (exac.broadinstitute.org), gnomAD (https://gnomad.broadinstitute.org), dbSNP (https://www.ncbi.nlm.nih.gov/SNP), and 1000 Genome Database (www.internationalgenome.org). This variant is classified as “likely pathogenic” according to the variant interpretation guidelines by the American College of Medical Genetics and Genomics.^[2]

Reverse transcription PCR (RT-PCR) products from the proband, proband's father, and mother were distinguished and identified a single band of 506 bp in proband's father whereas two bands of 506 bp and 328 bp in proband's mother and a band of only 328 bp in the proband [Figure 1C]. Direct sequencing of the RT-PCR products found that the 506-bp band was consisted of exons 47, 48, and 49 while the 328-bp band was comprised of only exons 47 and 49 [Figure 1D]. The splicing model was schematically described [Figure 1E]. Hence, this splicing variant causes a complete loss of exon 48 in the proband. Proband's father was harboring the wild-type (WT) COL4A5 transcript while proband's mother was identified with both WT transcript and the transcript with complete loss of exon 48 [Figure 1E].

COL4A5 gene is located in the long arm of chromosome X (Xq22.3) and translated to WT α5 chain of 1685 amino acids, with three domains, namely, signal peptide, Gly-X-Y repeats containing collagenous domain, short non-collagenous interruptions and a non-collagenous domain.^[1] Collagen α3, α4, and α5 monomers bind together with their NC1 domains and form a triple-helical structure which finally leads to the formation of a collagen network.^[3] This splicing variant can interfere with normal triple-helix formation.

Previously, three AS patients have been reported with variants causing skipping of exon 48. Knebelmann et al^[3] reported a male patient with c.4688G > A in exon 48 of the COL4A5 gene. The proband was manifested with hearing loss, maculopathy, and lenticonus, with ESRD at the age of 21 years. Weber et al^[4] reported a male patient with c.4688 + 1G > A in intron 48 of the COL4A5 gene. The proband has been suffering from hearing loss and ESRD. Wang et al^[5] reported a patient with c.4688 + 4G > A in intron 48 of the COL4A5 gene. The proband's detailed clinical manifestation was unknown. This finding may explain the phenotypic heterogeneity among the patients harboring the transcript with skipping of exon 48.

This study reported extreme phenotypic heterogeneity among three female patients with AS in this family. The affected females with a milder manifestation of disease identified with the WT transcript as well as the truncated transcripts. Hence, the phenotypic heterogeneity among these three female patients with AS in this family also supported the theory of random X-chromosome inactivation. AS patients are generally presented with ocular abnormalities including corneal opacities, anterior lenticonus, fleck retinopathy, and temporal retinal thinning, which were very helpful for the proper and timely clinical diagnosis, understanding of the likelihood of early onset renal failure, and mode of inheritance. However, unlike other AS patients, here, the proband was presented with astigmatism. Hence, our present finding expands the phenotypic spectrum for AS patients.

In conclusion, the present study identified a novel truncating splicing variant (c.4688 + 2T > C) in the COL4A5 gene causes XLAS in a four-generation Chinese family. This finding may provide new insights into the cause and diagnosis of AS.

Data availability statement

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Acknowledgements

We are thankful to the proband and all the family members for participating in our study.

Funding

This work was supported by a grant from the Shenzhen Key Medical Discipline Construction Fund (No. SZXK028).

Conflicts of interest

None.