Novel COL4A5 mutation in preimplantation genetic diagnosis of a patient with Alport syndrom

doi:10.21203/rs.3.rs-1425364/v1

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Novel COL4A5 mutation in preimplantation genetic diagnosis of a patient with Alport syndrom

https://doi.org/10.21203/rs.3.rs-1425364/v1

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Purpose We report a novel gene mutation in a female patient with Alport syndrome who was planning to undergo assisted reproduction to achieve pregnancy. We performed pathogenicity analysis on the mutation and selected a suitable assisted reproductive technology solution for the patient.

Methods A retrospective analysis of clinical and genetic data of a patient with Alport syndrome was performed. Whole-exome data of the COL4A5 gene of the patient’s family were analyzed using Sanger sequencing and online software was used to predict the conservation and pathogenicity of mutant amino acids within species evolution.

Results The patient planned assisted reproductive technology due to secondary infertility and unexplained hematuria. Urine test results revealed 60% severely deformed red blood cells, indicating persistent glomerular hematuria. Renal biopsy revealed that the basement membrane was of varying thickness, with a thickened dense layer, some of which was tear-like and arachnoid. Foot processes showed segmented fusion and the basement membrane was torn under electron microscopy. Genetic analysis revealed an unnamed mutation in the COL4A5 gene, c.3188G>A, which resulted in amino acid changes (p.Gly1063Asp). This was verified as a new mutation not present in the parents, whereas the daughter had a heterozygous mutation. Biological information analysis revealed the mutation was pathogenic and preimplantation genetic diagnosis was adopted. Eventually the patient gave birth to a full-term daughter without the gene mutation

Conclusion Genetic testing prior to pregnancy is recommended in Alport syndrome patients or family members prior to pregnancy to determine the specific location of the gene mutation. Patients should receive genetic counseling, fertility guidance, and assisted reproduction advice from experts in heredity.

Alport syndrome

COL4A5

gene mutation

genetic disease

preimplantation genetic diagnosis

Alport syndrome (AS) is the second most common renal genetic disease and is a phenotypic heterogeneous disease of the glomerulus, cochlea, and fundus membrane, which is mainly caused by mutations in the COL4A3, COL4A4, and COL4A5 type IV collagen genes (Miner, 1999; Kashtan, 1999) that encode the α3, α4, and α5 chains of type IV collagen, respectively. Type IV collagen consists of six different α-chains that interact to form three different heterotrimers (α1α1α2, α3α4α5, and α5α5α6) (Warady et al., 2020). Its structure is essential for the structural integrity and function of the glomerular basement membrane of the kidney, cochlea, and eye (Hudson, 2004; Pöschl et al., 2004),where it forms the basement membrane superstructure together with inline/nestin, laminin, and proteoglycan(Miner, 1999; Rheault, 2012). Consequently, patients with AS are at increased risk of renal failure, sensorineural hearing loss, and eye diseases(Watson et al., 2021).

The COL4A5, COL4A3, and COL4A4 genes have 53, 52, and 48 exons, respectively, and it is estimated that only 10% of all possible pathogenic COL4A5 variants have been reported (Gubler et al., 1981). Although more than 400 mutations have been reported in the COL4A5 gene, of which missense mutations account for 30%, there are no hotspot mutations. The α-5 (IV) chain of collagen is 1,685 amino acids in length and is expressed at 225 locations in the human glomerular basement membrane, retinal pigment layer, main visual cortex, and cochlea.

The disease characteristics of AS mainly include progressive glomerulonephritis, renal failure, sensorineural deafness, and specific ocular abnormalities (lenticular nucleus and macular spots). Abnormal α-chain structures destroy the integrity of the basement membrane, which initially leads to hematuria and later develops into moderate-to-severe proteinuria, progressive renal failure, high-pitched sensorineural hearing impairment, and eye disease. The pathology often manifests as thickness of the basement membrane of the kidney, delamination and rupture of the dense layer, damage to the anterior lens of the eye, cataracts, and macular degeneration (Kashtan, 2021; Jia et.al., 2020). Vision is usually unaffected in most patients with AS(Savige et al., 2015). AS remains a genetic disease that is often missed or misdiagnosed and its pathophysiology is still not fully understood, although some studies have reported that it may be related to chronic inflammation and metabolic dysfunction(Warady et al., 2020). There are three general modes of inheritance: X-linked dominant inheritance (X-linked Alport syndrome; XLAS), autosomal recessive inheritance, and autosomal dominant inheritance. XLAS due to a COL4A5 mutation in the Xq22 region accounts for about 80% of AS cases(Barua et al., 2018). In other cases, autosomal recessive inheritance caused by mutations in COL4A3 or COL4A4 accounts for around 15% of cases and autosomal dominant inheritance accounts for around 5% (Jia et al., 2020; Gao et al., 2020; Kruegel et al., 2013).

The clinical manifestations of XLAS in female patients are very different. Some patients have normal renal function and hearing, whereas others rapidly develop end-stage renal disease and deafness (Rheault, 2012). Around 1% of females with XLAS may also have tumors in the bronchus, esophagus, and vagina(Savige et al., 2016). Studies have shown that X-chromosome inactivation is a risk factor for disease progression in women with XLAS(Rheault, 2016).

Diagnosis of AS is mainly based on family history, clinical features, or pathological manifestations, such as renal biopsy, and finally diagnosed by genetic testing(Savige et al., 2016; Savige et al., 2013). Treatment methods include the use of hearing aids, hemodialysis, and peritoneal dialysis to treat end-stage renal failure and the last step is kidney transplantation. There is no specific and effective treatment method for AS(Gao, 2020), although the goal is to delay disease progression as much as possible and avoid renal failure (Kashtan, 2021). Studies have shown that treatment can temporarily delay the development of the disease. For example, antisense oligonucleotides can be used for exon skipping targeted therapy to improve the pathological manifestations and clinical symptoms of males with XLAS(Yamamura et al., 2020) or early treatment with angiotensin converting enzyme inhibitors(Kashtan, 2021). Individualized treatment for AS patients should be performed according to the identified gene mutation type, sex, and clinical stage of the disease (Kruegel et al., 2013). Next-generation high-throughput sequencing technology can identify up to 95% of pathogenic COL4A mutations, although some mutations caused by deep intron splicing or chimerism are difficult to detect(Savige et al., 2019). The development of sequencing technology has led to the discovery of more and more gene mutations.

In the field of assisted reproduction, preimplantation genetic testing (PGT) is defined as a test sample used to analyze a small amount of genetic material from oocytes (polar bodies) or embryos (cleavage or blastocysts) to determine whether there are genetic abnormalities and genetic aberrations, including preimplantation genetic diagnosis/screening before the revision. PGT is the preferred choice for most patients with infertility and genetic diseases.

Ethics and informed consent

The present study was approved by the Ethics Committee of the First Affiliated Hospital of Lanzhou University and the patient and their family members signed an informed consent form and agreed to give blood samples for genetic testing.

Clinical characteristics

The patient was a 36-year-old female (case II-3) weighing 56.5 kg, with a body mass index of 21 kg/m². Her daughter (case III-1) was delivered by cesarean section in 2016, and she experienced a spontaneous abortion in 2017. In May 2020, she was referred to the Assisted Reproductive Center of the First Affiliated Hospital of Lanzhou University due to being “married for 6 years and not pregnant without contraception.” A self-reported suspected AS was diagnosed at the local hospital in April 2019. Since 2018, the patient and her daughter have experienced unexplained intermittent hematuria, nocturia, and fatigue. The patient was admitted to hospital and underwent hysterosalpingography, which showed that both fallopian tubes were unobstructed. Basic blood hormones levels revealed thyroid-stimulating hormone levels of 1.375 ulU/mL and anti-Müllerian hormone levels of 1.223 ng/mL. There were 10 antral follicles. Blood tests revealed white blood cell count of 10.7 × 10⁹/L; hemoglobin, 155 g/L, hematocrit of 44.9%, average hemoglobin content of 32.8 pg, and 78.7% neutrophils. Routine urine examination showed positive occult blood (++), 72 red blood cells/μL, and 41 abnormal red blood cells/μL. The urine test results of the patient’s daughter showed occult blood (+++), red blood cell count of 2,104.1, red blood cell count of 378/μL, no sporulated red blood cells, 30% circular red blood cells, 30% perforated red blood cells, and 30% other deformed red blood cells. Severely deformed red blood cells accounted for 30%. Both the patient and her daughter were considered to have glomerular hematuria. The pathological results of kidney puncture in case III-1 are shown in Figure 1. Multifrequency steady-state auditory evoked potentials and fundus examinations showed that the patient and her daughter had no high-frequency hearing impairment and no changes in retinopathy. Thus, there were no abnormal changes in hearing and vision. No other family members showed clinical symptoms, such as hemorrhage, proteinuria, hearing loss, or eye diseases.

Methods

COL4A5 gene amplification

Genetic testing should be performed for individuals with suspected AS (Gao et al., 2020). In order to clarify the diagnosis, 2 mL of peripheral blood was drawn from the patient and other family members for genetic testing after the review by the hospital ethics committee and obtaining informed consent of the patient and family members. DNA was extracted from peripheral blood, and genomic DNA was extracted using blood genomic column small-amount extraction kit (CWbio, CW2087). The genomic DNA sequence of COL4A5 was obtained from the GenBank database and Primer5 software was used to design a primer sequence that could specifically amplify the gene encoding exon and part of the introns on both sides of the exon using 2× Gold Star Best Master Mix (CWbio, CW0655M), genomic DNA of each individuals as a template, and COL4A5-specific amplification primers. Target gene amplification was performed using a TC-XP-G polymerase chain reaction (PCR) machine (BIOER), with a 30-s extension to obtain PCR product sequencing.

DNA sequencing analysis of COL4A5 gene

DNA sequencing was performed using the purified PCR product as a template on a PCR machine using a DNA cycle sequencing kit and a COL4A5 gene-specific amplification primer. PCR products were purified uing a QIAquick purification kit (Qiagen, Germany) and then loaded onto a DNA sequencer (Applied Biosystem, USA) for sequencing. The detected COL4A5 sequence was compared with the COL4A5 sequence in the Nucleotide database (https://www.ncbi.nlm.nih.gov/Nucleotide) to detect the COL4A5 gene variation. Searches in Clinvar, HGMD, PubMed, and GeneReview were conducted to determine whether the COL4A5 gene mutation found had been previously reported.

Theoretical pathogenicity analysis

Theoretical pathogenicity analysis was carried out according to the interpretation standards and guidelines of gene mutations compiled by the American Society of Medical Genetics and Genome and the American Society of Molecular Pathology (AMP). Specific standard terms including “pathogenic,” “probably pathogenic,” “probably benign,” “benign,” and “unclear” were used to describe the variants found in Mendelian disease(Richards et al., 2015). The COL4A5 gene c.3188G>A mutation is a new mutation and none of the parents carried this mutation. The patient and her daughter carried a mutation that conformed to the X-linked genetic characteristics (strong evidence of pathogenicity, PS2).

A search of the Thousand Human Genome Database and EXAC Database revealed that no healthy person carried the c.3188G>A (medium pathogenicity evidence, PM2). The occurrence of c.3188G>T, p.Gly1063Val at the same position, pathogenic, was a different missense mutation of pathogenicity (medium pathogenicity evidence, PM5). The missense variant of COL4A5 is the cause of AS and the proportion of benign variants in this gene is 0.00069 (dbSNP) (supporting pathogenicity evidence, PP2). The clinical manifestations of the patient and the affected family members were consistent with the suspected diagnostic phenotype of AS, and other related diseases were excluded (supporting pathogenicity evidence, PP4). In summary, the strength of evidence for this mutation is “PS2 + PM2 + PM5 + PP2 + PP4,” which was theoretically determined as a pathogenic mutation.

Bioinformatics analysis

Bioinformatics online websites were used to predict the effect of the mutation on the function of the protein and the strength of the pathogenicity was performed using PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/index.shtml), PROVEAN (http://provean.jcvi.org/index.ph), SIFT (http://sift.jcvi.org/), MutationTaster (http://www.mutationtaster.org/), FATHMM (http://fathmm.biocompute.org.uk), CADD (http://cadd.gs.washington.edu), Mutation Assessor (http://www.mutationassessor.org), and M-CAP (bejerano.stanford.edu/MCAP).

The Clustar omega was predicted using the website (https://www.ebi.ac.uk/Tools/msa/clustalo) and the human collagen type IV is was found to be different from that of canines, mice, zebrafish, chimpanzees, pigs, and cats. The COL4A5 protein sequence of the species was compared and estimated for conservation. In phastCons-vertebrate (http://compgen.bscb.cornell.edu/phast/) and PhyloP Vertebrate (http://compgen.bscb.cornell.edu/phast/) conservatively predicts this site in humans.

Prediction of splicing site changes was performed using MutationTaster.

Genetic testing

The results of the genetic testing showed that the patient (cases II-3) and her daughter (cases III-1) had a heterozygous mutation c.3188G > A (missense mutation) in COL4A5 gene at chrX: 107869521 (NM_000495.5 exon36), resulting in an amino acid change p.Gly1063Asp, and the disease was inherited by X-linked dominant inheritance. The genetic map of this family is shown in Fig. 2. The mutation was further confirmed by Sanger sequencing (Fig. 3).

The discovered COL4A5 gene mutations were queried in using mutation databases, such as dbSNP (https://www.ncbi.nlm.nih.gov/projects/SNP/) and 1000 Genome Project (ftp://ftp- trace.ncbi.nih.gov/1000genomes/ftp/release), and it was found that the mutation has never been reported. A novel heterozygous variant in the COL4A5 gene was identified.

Other relevant examinations of the patient did not reveal eye or ear damage. We analyzed and discussed this case with nephrologists. A decision was made to use the intracytoplasmic sperm injection (ICSI) regimen for assisted reproduction, and since there was no effective treatment for this genetic disease, we gave the patient symptomatic treatment. Finally, 8 eggs were collected, 3 transferable blastocysts were obtained by culture, and 1 normal embryo was obtained by PGT test. Embryo transfer was performed after consultation and informed consent of the patient's couple. One daughter was born alive at term after transplantation.

Bioinformatics analysis

Prediction results for the pathogenicity of the mutation using different biological information websites indicated that the site mutation may be pathogenic. The phastCons-vertebrate website indicated that the site and its nearby sequences were conserved and the PhyloP Vertebrate website indicated that the site was conserved (Table 1).

Table 1

The variant c.3188G > A (p. Gly1063Asp) in COL4A5 (NM_000495.5) annotated by ANNOVAR.
Gene	COL4A5
Position	ChrX (hg19): 107869521
Single nucleotide variant	c.3188G > A, p.Gly1063Asp
Polyphen2	Probably damaging
PROVEAN	Deleterious
SIFT	Deleterious
Mutation Taster	disease causing
FATHMM	Damaging
CADD	Deleterious
Mutation Assessor	Deleterious
M-CAP	Probably damaging
phastCons-vertebrate	Conserved
PhyloP Vertebrate	Conserved
Polyphen2 score, 0.99 (scores closer to 1 indicate a higher pathogenicity of the mutation); PROVEAN score, − 5.733; and threshold, − 2.5 (smaller values indicate a higher pathogenicity). CADD score, 28.0 (the threshold is 20.0). Mutation Assessor predicts the level of impact on protein function, which was high. M-CAP score, 0.981 (threshold value, 0.025); phastCons-vertebrate score, 5.658 (threshold value, 0); and PhyloP Vertebrate score, 1 (scores closer to 1 indicate they are more conservative).

Alignment analysis showed that the amino acid G at position 1,063, corresponding to COL4A5 genes of the seven species, was conserved (Fig. 4). The COL4A5 gene of humans and chimpanzees is earlier in evolution time than that of other species, and the zebrafish COL4A5 gene is the latest in evolution time. The evolutionary lineage changes over time. The longer the evolution length, the greater the changes in the corresponding species’ genes. Therefore, in the evolutionary branches of humans and chimpanzees, changes in this gene are relatively conservative (Fig. 5).

Changes in splicing sites were analyzed. In order to reduce the number of false-positive splicing site predictions, Loss/decrease of splice sites distant from intron-exon (and reverse) borders will be ignored. Mutation Taster only considers the loss or strength of splicing sites on the existing intron–exon boundary.This will be taken for a "real" splice site change. The results showed that there were three splice sites enhanced and one additional splice site activated (Table 2). That is, the normal splicing site was affected.

Table 2

Prediction of splicing site
Effect of Splice site	gDNA position	score	wt detection sequence	exon-intron border
Donor increased	186440	wt:0.49 mu:0.62	wt: CCTGGACAGAAAGGC mu: CCTGGACAGAAAGAC	TGGA\|CAGA
Donor gained	186444	0.59	mu: GACAGAAAGACGACA	CAGA\|AAGA
Donor increased	186446	wt:0.58 mu:0.70	wt: CAGAAAGGCGACAAA mu: CAGAAAGACGACAAA	GAAA\|GGCG
Donor increased	186449	wt:0.87 mu:0.98	wt: AAAGGCGACAAAGGT mu: AAAGACGACAAAGGT	AGGC\|GACA
MutationTaster determines the position of this splice site change relative to intron/exon borders. An increase in an existing splice site is displayed if the change in the confidence score is > 10% [splice site became stronger (increased) or weaker (decreased), additional splice site activated (gained) or splice site completely lost]. A gain of a completely new splice site was displayed if the confidence score of the newly created splice site was > 0.3.

The present study identified the c.3188G > A (p.Gly1063Asp) mutation in the COL4A5 gene in two members of a Chinese family. This is a novel gene mutation and the pathological findings of the kidney showed tearing of the basement membrane. Exome sequencing of the X-chromosome revealed that the patient and daughter gene COL4A5 gene (exon36) had a missense mutation c.3188G > A (missense mutation, p.Gly1063Asp). This mutation has not been described in any public single nucleotide polymorphism database. The entire family was investigated and analyzed. The proband was a woman who intended to undergo assisted reproduction and genetic sequencing found that both the patient and her daughter had mutations in the gene locus, whereas the other family members were normal. To date, the two patients have not experienced renal failure, hearing impairment, or eye disease and it is important to delay disease progression and closely monitor the condition.

The patient was diagnosed with AS and her husband had oligoasthenospermia. We intended to help her to become pregnant via PGT. We used the long-term luteal phase program to promote ovulation for 12 days. Under the guidance of vaginal ultrasound, a 17 G single-cavity puncture needle was sucked under negative pressure of − 120 mmHg and both ovaries were punctured through the vaginal fornix. A total of six eggs were collected. The husband’s semen assessment showed a sperm concentration of 2–4/20 HP, of which there were 1–2 motile sperm/20 HP and 1–2/HP nonmotile sperm. Four normal fertilized embryos were obtained via ICSI, all of which were high-quality, and four normal fertilized high-quality embryos were obtained via ICSI technology. Two transferable embryos were finally obtained after the embryos were sent for examination. However, transplant was canceled due to the work commitments of the patient. After discussing with the patient, we planned to determine the right time to transfer the embryo into the patient's uterus.

Accurately distinguishing between neutral mutations and pathogenic mutations is greatly significant to the clinical detection of genetic diseases. We immediately made a mutation prediction for the mutation as multiple sites predicted that the mutation was pathogenic and may cause changes in protein function. The multiple sequence alignment of COL4A5 protein sequences of different species and human type IV collagen highlights the high evolutionary conservation of glycine-1,063. There are few limitations to this study. First, we only made a functional prediction of the pathogenicity of the new mutation and did not conduct further experimental studies. The number of case studies was insufficient to generalize all results, and the conclusions from this study were difficult to effectively apply to mutations at other sites. However, we believe that more mutations will be reported.

The mutation site in XLAS patients has not been reported. The newly discovered COL4A5 mutation supplements the human AS gene mutation database, which is significant for further studies on the pathogenesis of AS, genetic counseling, and prenatal diagnosis. This is the novel insight. Previous studies have shown that most pediatric patients diagnosed with AS have atypical clinical manifestations from their parents and it is difficult to detect AS patients in time(Vos et al., 2018); therefore, nephrologists should focus on the emergence of patients with unexplained hematuria, proteinuria, or renal failure and determine whether there is a positive family history of kidney disease. Patients with XLAS are not uncommon in clinical practice, with around one case of X-linked AS patients in every 50,000 newborns(Vos et al., 2018). There is still no cure and the prognosis is poor and often progresses to end-stage renal failure. Most of the initial clinical manifestations of AS patients are not typical, making it difficult to identify these patients during the early stage. Therefore, once patients with suspected AS are diagnosed with the syndrome, family genetic investigation and genetic testing should be carried out as soon as possible. Eye examinations are recommended at least once a year for female XLAS patients who may experience hearing loss in middle-age and they should be checked regularly if they suffer from renal failure (Tsang and Sharma, 2018). Genetic testing before pregnancy is recommended for AS patients or their family members who would like to have children as it is important to determine the specific types and locations of any gene mutations, screen high-risk family members, and obtain appropriate genetic counseling and fertility guidance from a specialist with clinical genetics knowledge based on their inheritance to reduce the incidence of AS. Special attention should be also paid to pregnancy complications caused by renal damage and hypertension in patients who have succeeded in assisted reproduction. Further studies are required to clarify the disease progression of AS and it is hoped that a better understanding can lead to better control and a cure.

Ethics Statement

This study was approved by the Ethics Committee of the First Affiliated Hospital of Lanzhou University, and the patient and family members have signed an informed consent form and agreed to take blood for genetic testing. Written informed consent was obtained from the patient for publication of this case report and any accompanying images.

Author Contributions

RH and XM Ma participated in experiments and wrote this article. AS, BM and XS were responsible for sample collection and information collection. YY guided the entire essay

Funding

None.

Acknowledgments

Thanks to the members of the family for their help and participation in the study.

Disclosure Statement

The authors have no conflicts of interest to declare.

Data Statement

None

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Novel COL4A5 mutation in preimplantation genetic diagnosis of a patient with Alport syndrom

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