Effect of heterozygous pathogenic COL4A3 or COL4A4 variants on patients with X-linked Alport syndrome.

BACKGROUND: Alport syndrome is an inherited renal disease caused by mutations in COL4A3, COL4A4, or COL4A5 genes. Coexisting mutations in either two of the three genes in Alport patients have been reported recently. However, the effect of heterozygous mutations in COL4A3 or COL4A4 genes in X-linked Alport syndrome (XLAS) patients is unclear.
METHODS: Using targeted next-generation sequencing, six unrelated Chinese children were identified to have a combination of a pathogenic variant in COL4A5 and a heterozygous mutation in COL4A3 or COL4A4. They were three males and three females. Another three XLAS males each with only one pathogenic variant in COL4A5 were included. The clinical data were analyzed and compared between the males in two groups (group 1, males with a pathogenic variant in COL4A5 and a heterozygous pathogenic variant in COL4A3 or COL4A4; group 2, males with only one pathogenic variant in COL4A5).
RESULTS: Patients with XLAS who also had heterozygous pathogenic COL4A3 or COL4A4 variants accounted for 1% of Alport syndrome. In this study, three children showed coexisting pathogenic variants in COL4A5 and COL4A3. Two children showed pathogenic variants in COL4A5 and COL4A4. One child had pathogenic variants in the three COL4A3-5 genes, in which the pathogenic variant in COL4A5 was de novo and the pathogenic variants in COL4A4 and COL4A3 were inherited independently (in trans). The site and type of mutations in COL4A5 were similar between the two groups. It was revealed that males in group 1 presented more severe proteinuria than males in group 2 (p < 0.05).
CONCLUSION: The present study provides further evidence for complicated genotype in Alport syndrome. For the first time, we reported a case with three pathogenic variants in COL4A5, COL4A3, and COL4A4 genes. Moreover, we found that heterozygous pathogenic COL4A3 or COL4A4 variants are likely to make XLAS disease more serious.

INTRODUCTION

Alport syndrome is a hereditary renal disease characterized by hematuria, proteinuria, progressive renal failure, and frequently with hearing loss or ocular abnormalities. The typical ultrastructural changes of kidney in Alport syndrome are diffuse glomerular basement membrane (GBM) lamellation (Flinter, 1997; Kashtan, 1999; Zhang & Ding, 2018). Alport syndrome is caused by mutations in the COL4A3 (OMIM, # 120070), COL4A4 (OMIM, # 120131), or COL4A5 (OMIM, # 303630) genes encoding collagen IVα3, α4, and α5 chains (Barker et al., 1990; Gross, 2008; Mochizuki et al., 1994). About 85% of individuals with Alport syndrome are X‐linked inherited due to mutations in the COL4A5 gene. Males with X‐linked Alport syndrome (XLAS) are affected more severely than females (Massella et al., 2003; Raju, Cimbaluk, & Korbet, 2013; Savige, Colville, et al., 2016; Savige, Storey et al., 2016). Ninety percent of males with XLAS progress to end‐stage renal disease (ESRD) by age 40, but only about 20% of females with XLAS develop renal failure by age 60 (Jais et al., 2000, 2003; Naito, Kawai, Nomura, Sado, & Osawa, 1996; Wang, Ding, Guo, & Yang, 2002; Wang et al., 2012; Yamamura et al., 2017). Fifteen percent of individuals with Alport syndrome are autosomal recessive inherited caused by homozygous or compound heterozygous mutations from both alleles of either COL4A3 or COL4A4 genes (Storey, Savige, Sivakumar, Abbs, & Flinter, 2013; Wang et al., 2014; Zhang et al., 2012). Both males and females with autosomal recessive Alport syndrome (ARAS) have a high risk of ESRD by age of 30 (Kashtan et al., 2013; Oka et al., 2014).

However, the phenotype of individuals with heterozygous mutations in COL4A3 or COL4A4 genes varies widely. In our previous study, parents of ARAS children who were carriers of heterozygous mutations in COL4A3 or COL4A4 genes, 53% of them had normal urinalysis, 31% had hematuria, and 16% had hematuria and proteinuria (Zhang et al., 2012). The diseases associated with heterozygous mutations in COL4A3 or COL4A4 genes are thin basement membrane nephropathy and autosomal dominant Alport syndrome (Fallerini et al., 2014; Kamiyoshi et al., 2016; Savige et al., 2003).

To date, the effect of heterozygous mutations in COL4A3 or COL4A4 genes in XLAS patients is unclear. It is important to know whether it would make the XLAS disease worse. Here, we reported six unrelated Chinese children with XLAS who were also detected with heterozygous mutations in COL4A3 or COL4A4 genes. Our study aimed to provide more information on clinical assessment and genetic counseling for Alport syndrome.

MATERIALS AND METHODS

Patients and families

Patients diagnosed or suspected of Alport syndrome in the department of Pediatrics, Peking University First Hospital from 2014 to 2017 were screened for mutations in the COL4A3, COL4A4, COL4A5, and COL4A6 genes. The clinical diagnosis criteria included ① glomerular hematuria, proteinuria, or renal failure, ② family history of Alport syndrome or renal failure without other exact disease, ③ lack or discontinuous staining of α5 (IV) chain in epidermal basement membrane (EBM), or in GBM, ④ the GBM lesions under electron microscopy (irregular thinning, thickening with splitting, and lamellation), ⑤ one pathogenic mutation in COL4A5 or two pathogenic mutations in COL4A3 or COL4A4 genes. Alport syndrome was suspected for individuals with criteria ① and ②. Alport syndrome was diagnosed for individuals with criteria ① ② and one of ③ ④ ⑤.

Six unrelated XLAS children were found to have pathogenic variants in COL4A5 gene and also have heterozygous pathogenic variants in COL4A3 or COL4A4 genes. They were three males and three females. Then another group of XLAS patients was enrolled in this study according to the following criteria: ① with only one pathogenic variant in COL4A5 gene, ② with frameshift variant in exon 37, large deletion in exon 42, or glycine substitution in exon 25 of COL4A5 gene, ③ male. Finally, there were three males of XLAS enrolled. Therefore, there were two groups of male patients (group 1, males with a pathogenic variant in COL4A5 and a heterozygous pathogenic variant in COL4A3 or COL4A4; group 2, males with only one pathogenic variant in COL4A5). For all subjects, clinical data including gender, age of disease onset, initial symptoms, extrarenal manifestations, results of skin biopsy and renal biopsy, family history, therapy, kidney function at latest follow‐up were collected.

Ethical compliance

The Ethical committee of Peking University First Hospital approved the project, and informed consent was obtained from the probands and their family members.

Genomic DNA and targeted NGS

A sample of peripheral blood in EDTA tubes was collected from probands and all available family members. Gnomic DNA was isolated from the blood samples using a FlexiGene DNA Kit (Qiagen) according to the manufacturer's protocol. Gnomic DNA from probands was detected for mutations in COL4A3‐6 genes. Targeted next‐generation sequencing (NGS) was performed by BGI‐Tianjin, China as published previously (Wang et al., 2017). The pathogenicity of variants identified was based on meeting at least one of the following criteria: (a) truncating mutations (nonsense, consensus splice site ±1 or 2 nucleotide, large deletion, and frameshift), (b) variants previously described as disease causing in a patient with a similar phenotype in the website (HGMD, LOVD, and Clin Var), (c) Glycine missense variants in the intermediate collagenous domains (except p.Gly624Asp in COL4A5), or (d) novel non‐Glycine substitutions absent or at very low frequency in large population cohorts (1,000 genomes; ExAC, gnomAD), in domain high evolutionary conservation, and more than two prediction scores classified the allele as disease causing (SIFT, Mutation Taster, Polyphen 2) (Richards et al., 2015; Savige et al., 2018). The pathogenic mutations identified with NGS in probands were confirmed by Sanger sequencing or qPCR (real‐time quantitative PCR) for large deletion mutations. Gnomic DNA from family members was analyzed by Sanger sequencing or qPCR to determine whether they had the same mutations as the probands.

Statistical analysis

Statistical analysis was performed by SPSS 19.0. The differences of proteinuria level, age, and time of therapy in two groups were compared using unpaired t test. If the p value < 0.05, the differences were considered to be statistically significant.

RESULTS

In this study, among 417 patients diagnosed or suspected of Alport syndrome in our department during 2014–2017, six were identified with pathogenic variants in COL4A5 plus heterozygous pathogenic variants in COL4A3 or COL4A4, which accounted for 1%. They were three males (proband 1, 2, 3) and three females (proband 4, 5, 6). Another three males (proband 7, 8, 9) with only one pathogenic variant in COL4A5 were included. The sites and types of pathogenic variants in COL4A5 gene were similar between the two groups of males. Two frameshift mutations in exon 37 p.G1110Afs*45 and p.G1098Vfs*54 in COL4A5 gene were detected in proband 1 and proband 7, respectively. The same large deletion mutation (deletion of exon 42) in COL4A5 gene was detected in both proband 2 and proband 8. Two missense mutations p.G594R and p.Gly644Val in exon 25 in COL4A5 gene were detected in proband 3 and proband 9, respectively. The pathogenic variants identified in these children were shown in Table 1.

Table 1

Pathogenic variants in COL4A3‐5 genes identified in nine children with X‐linked Alport in this study

Proband number	Gender	Gene	Exon (intron)	Variant	Effect on protein	Type	dbSNP ID	SIFT (scroe)	PolyPhen2 (scroe)	Mutation Taster	Reference	Variant derived
												Father	Mother
1	M	COL4A5	37	c.3328_3329insCAAACCAG	p.G1110Afs*45	Frameshift	—	—	—	DC	LOVD	—	Het
		COL4A3	47	c.4207G>A	p.G1403R	Missense	rs772528863	D(0)	PD(1)	DC	LOVD	—	N
2	M	COL4A5	42	Exon42 del	—	Exon deletion	—	—	—	—	—	—	—
		COL4A3	44	c.3946G>A	p.G1316S	Missense	—	D(0)	PD(1)	DC	LOVD	—	N
3	M	COL4A5	25	c.1780G>C	p.G594R	Missense	rs104886131	D(0)	PD(0.901)	DC	—	—	Het
		COL4A4	48	c.4915G>C	p.G1639R	Missense	rs749899964	D(0)	PD(1)	DC	LOVD	—	Het
4	F	COL4A5	1	c.50delT	p.L17Rfs*27	Frameshift	—	—	—	DC	—	N	N
		COL4A3	50	c.4664C>T	p.A1555V	Missense	rs369575989	D(0)	PD(1)	DC	—	N	Het
		COL4A4	32	c.2932G>A	p.G978R	Missense	rs759439914	D(0)	PD(1)	DC	—	Het	N
5	F	COL4A5	35	c.3094_3095delAT	p.M1032Gfs*35	Frameshift	—	—	—	DC	—	N	N
		COL4A3	32	c.2549G>A	p.G850E	Missense	—	D(0)	PD(1)	DC	—	Het	N
6	F	COL4A5	(47)	c.4511‐2A>G	—	Splicing	—	—	—	—	—	—	Het
		COL4A4	41	c.3826G>A	p.G1276R	Missense	—	D(0)	PD(1)	DC	—	—	Het
7	M	COL4A5	37	c.3293delG	p.G1098Vfs*54	Frameshift	—	—	—	DC	—	—	Het
8	M	COL4A5	42	Exon42 del	—	Exon deletion	—	—	—	—	—	—	—
9	M	COL4A5	25	c.1931G>T	p.Gly644Val	Missense	—	D(0)	PD(1)	DC	—	—	—

Pathogenicity of missense variants predicted using SIFT, MutationTaster, and PolyPhen‐2.

The version number of COL4A3, COL4A4, or COL4A5 gene was NM_000091.4, NM_000092.4, and NM_033380.2, respectively.

D: deleterious; PD: probably damaging; DC: disease causing; N: normal; Het: heterozygous; LOVD: Leiden open variation database.

The clinical manifestations were compared between the two groups of XLAS males (Table 2). All the boys in the two groups had initial symptoms of hematuria and proteinuria, and positive family history of kidney disease. The onset age of the disease, and hearing loss rate and age had no difference between the two groups. Kidney biopsy information was available in four boys. Skin biopsy and α5 (IV) chain staining in the EBM were underwent in three boys and two of their mothers. Negative staining of collagen IV α5 chain was identified in either EBM or GBM in five boys. All the six boys had angiotensin‐converting enzyme inhibitor (ACEI) and angiotensin receptor blocker (ARB) for treatment after diagnosis. The amount of ACEI and ARB for each patient was 0.25 mg−1kg−1d and 0.5 mg−1kg−1d, respectively. The length of time for therapy and the age at latest follow‐up had no difference between the two groups. All the boys had normal renal function at latest follow‐up. However, the proteinuria during follow‐up was more severe in three boys (proband 1, 2, 3) with a combination of one pathogenic variant in COL4A5 and one heterozygous pathogenic variant in COL4A3 or COL4A4. And the difference between the two groups was statistically significant (p < 0.05).

Table 2

Comparison of clinical manifestations of two groups of males with X‐linked Alport syndrome

Proband number	Sex	Onset age (years)	Initial symptom	Family history of kidney diseases	Extrarenal manifestations (age, years)	α5 (IV) chain staining in the EBM	Renal biopsy(age, years)		Treatment (onset age, years)	Latest follow‐up
							Electron microscope findings	α5 (IV) chain staining		Age (years)	Urinary RBC/HP	24‐hr urinary protein (g/d)	Ccr(ml/1.73m2/min)	Scr (umol/L)
1	M	11.8	Hematuria, proteinuria	Yes	Hearing loss(6)	ND	Thinning, thickening and splitting, basket‐weaving of GBM(11.8)	Negative staining of α5 (IV) chain in GBM, BC, TBM	ACEI and ARB (11.8)	11.8	15	3.08	132.6	36.3
2	M	12	Hematuria, proteinuria	Yes	Hearing loss(14)	ND	ND	Negative staining of α5 (IV) chain in GBM, BC, TBM (13)	ACEI and ARB (12)	14	5	3.65	189.5	68
3	M	5	Hematuria, proteinuria	Yes	ND	Negative; discontinuous staining (the mother)	ND	ND	ACEI and ARB (5)	16	ND	1.63	145.21	65.9
7	M	11.3	Hematuria, proteinuria	Yes	Hearing loss(11)	ND	ND	ND	ACEI and ARB (11)	11	40–50	0.59	116.41	54.5
8	M	2.3	Hematuria, proteinuria	Yes	Hearing loss(12)	Negative; discontinuous staining (the mother)	Thinning of GBM(2)	Negative staining of α5 (IV) chain in GBM, BC, TBM	ACEI and ARB (3)	13	50–100	0.47	97.24	59.5
9	M	9	Hematuria, proteinuria	Yes	Hearing loss(9)	Negative	Thinning, thickening and splitting of GBM (14)	ND	ACEI and ARB (14)	14	20–30	1.17	138	74

Group 1: proband 1, 2, 3 (males with one pathogenic variants in COL4A5 and one heterozygous pathogenic variant in either COL4A3 or COL4A4); Group 2: proband 7, 8, 9 (males with only one pathogenic variant in COL4A5).

EBM: epidermal basement membrane; GBM: glomerular basement membrane; BC: Bowman's capsule; TBM: tubular basement membrane; ACEI: angiotensin‐converting enzyme inhibitor; ARB: angiotensin receptor blocker; α5 (IV): collagen IV α5 chain; Ccr: creatinine clearance rate; Scr: serum creatinine concentration; ND: no data.

In the three females (proband 4, 5, 6), the initial symptom was hematuria and the onset age was from 5 months to 3.6 years (Table 3). During follow‐up, proteinuria was presented in proband 6 at 5 years and was 0.207 g/d at 8 years under ACE inhibitor therapy. Proband 4 was a 3.6‐year‐old girl with three pathogenic variants in COL4A5, COL4A3, and COL4A4. She had no family history. Hearing was not detected. Renal biopsy at 3.6 years showed thinning and thickening of GBM. She was presented with hematuria and proteinuria at last follow‐up (3.8 years).

Table 3

Clinical manifestations of three females with complicated genotype

Proband number	Sex	Onset age (years)	Initial symptom	Family history of kidney diseases	Extrarenal manifestations (age, years)	α5 (IV) chain staining in EBM	Renal biopsy (age, years)	Treatment (onset age, years)	Latest follow‐up
									Age (years)	Urinary RBC/HP	24‐hr urinary protein (g/d)	Ccr (ml/1.73m2/min)	Scr (Umol/L)
4	F	3.6	Hematuria	No	ND	ND	Thinning and splitting of GBM (3.6)	No	3.8	100–120	a	NA	NA
5	F	5 months	Hematuria	Yes	ND	ND	ND	No	4	ND	0.12	NA	33.9
6	F	2	Hematuria	Yes	ND	ND	Thinning and thickening of GBM (4)	ACEI(5)	8	Full visual field	0.207	NA	33

EBM: epidermal basement membrane; GBM: glomerular basement membrane; ACEI: angiotensin‐converting enzyme inhibitor; Ccr: creatinine clearance rate; Scr: serum creatinine concentration; ND: no data.

urinary protein creatinine ratio 0.32 g/g.

Family segregation analysis was performed in all available family members. In proband 1 and proband 2, only their mothers’ samples were available and it revealed the pathogenic variants in COL4A5 were inherited from the mother, but the pathogenic variants in COL4A3 were not from the mother. In proband 3 and proband 6, it revealed that both of the pathogenic variants in COL4A5 and COL4A4 genes were inherited from the mother. And the pathogenic variants in COL4A5 gene were de novo in proband 4 and proband 5. In addition, the two heterozygous pathogenic variants in COL4A3 and COL4A4 genes in proband 4 were inherited in trans (on opposite chromosomes) from the mother and the father, respectively. The pedigrees of the six families were shown in Figure 1.

Figure 1

Pedigrees of six families presenting with mutations in more than one of three COL4A3‐5 genes. Proband is indicated by an arrow. Square indicates male; circle, female; black symbol, individual with clinical symptoms

DISCUSSION

The present study provides further evidence for complicated genotype in Alport syndrome. About 1% of patients diagnosed or suspected for Alport syndrome had mutations in more than one of the COL4A3‐5 genes. For the first time, we reported a case with three pathogenic variants in COL4A5, COL4A3, and COL4A4 genes. Moreover, we found initially XLAS males with an additional heterozygous pathogenic COL4A3 or COL4A4 variant presented heavier proteinuria than the XLAS males with only one pathogenic variant in COL4A5.

So far, only two males with pathogenic variants in COL4A5 and COL4A4 genes have been reported (Fallerini et al., 2017; Mencarelli et al., 2015). One was 26 years old with variant p.Gly1348Arg in COL4A5 and heterozygous variant p.Gly722Ser in COL4A4 presented with hematuria and proteinuria. Another male with variant p.Gly684Val in COL4A5 and heterozygous variant p.Pro1587Arg in COL4A4 had ESRD. The onset age of ESRD was not recorded. In addition, three females with combination of a pathogenic variant in COL4A5 and a heterozygous pathogenic variant in COL4A3 or COL4A4 had been reported. Their age was 9, 45, and 54 years old. The 9‐year‐old female was presented with hematuria and proteinuria. The 45‐year‐old female was presented with hematuria, proteinuria, and hearing loss. The 54‐year‐old female had ESRD at the age of 44. Therefore, these reported data from adult patients were consistent with our results that an additional heterozygous pathogenic COL4A3 or COL4A4 variant would make XLAS disease worse.

There were patients reported to have a combination of pathogenic variants in COL4A3 and COL4A4 (Fallerini et al., 2017; Kashtan et al., 2018; Mencarelli et al., 2015). In some cases the variants were on the same homologous chromosome (in cis). But in other cases the variants were inherited independently (in trans), like proband 4 in our study. Learning from the reported data, individuals with two heterozygous pathogenic variants in COL4A3 and COL4A4 in trans had more severe phenotype than those with a single heterozygous pathogenic variants, but had less severe phenotype than ARAS. However, there was no reference about XLAS patients combined with two heterozygous pathogenic variants in COL4A3 and COL4A4 in trans. The proband 4, a 3.6‐year‐old girl, reported here is the first case with this kind of complicated genotype. It is unknown whether her phenotype would be more severe than females with XLAS or ARAS. The long‐term follow‐up and more cases in the future might let us know more.

Besides, we should be alert that the three XLAS males with heterozygous pathogenic COL4A3 or COL4A4 variant in this study showed negative staining of α5 (IV) chains in EBM or GBM. A clinical diagnose of XLAS is definite (Hashimura et al., 2014; Savige et al., 2013; Wang et al., 2012; Wei et al., 2006). However, the genotype may be various, which would affect the risk of inheritance in families (Artuso et al., 2012; Kashtan et al., 2018). Therefore it is necessary to test all the three genes COL4A3‐5 by NGS in patients suspected for Alport syndrome, not only meaningful for diagnosis, but also for the genetic counseling (Gross et al., 2017; Kashtan et al., 2018; Savige, Colville, et al., 2016).

In conclusion, we reported six XLAS children with heterozygous pathogenic COL4A3 or COL4A4 variants, which accounted for 1% of Alport syndrome. One of them was the first case with three pathogenic variants in COL4A5, COL4A3, and COL4A4 genes. Our data revealed an additional heterozygous pathogenic COL4A3 or COL4A4 variant would make XLAS males suffering from more severe proteinuria. It suggested that another genetic hit from COL4A3 or COL4A4 might make the XLAS disease worse.

CONFLICT OF INTEREST

The authors have no conflict of interest to declare.