Long-term follow-up of an Alport syndrome patient with a novel mutation of COL4A5

Abstract

Background: Alport syndrome (AS) is a genetic disease characterized by progressive glomerulonephritis with a high life-time risk for end-stage renal disease (ESRD), sensorineural hearing loss and ocular abnormalities. So far, a lot of mutations were reported in COL4A3, COL4A4 and COL4A5 genes, which are related to AS. Methods: Whole-exome sequencing in combination with AS-related genes filtering strategy was applied to investigate a Chinese AS family. We also employed Sanger sequencing to confirm the family co-segregation. In addition, we also summed up a long-term follow-up data from 2003 to 2016. Results: In this study, we have detected a novel insertion mutation (c.348_349insTCCGG/p.G117Sfs×40) of COL4A5, which may lead to a truncated protein in the proband. Sanger sequencing confirmed that this novel mutation was co-segregated with all the family members. The long-term follow-up data showed that the progress of chronic kidney disease become more and more serious in the proband. Conclusions: A novel mutation (c.348_349insTCCGG/p.G117Sfs×40) of COL4A5 was identified in this study. In addition, approximately 15 years long-term follow-up data was provided in this paper. Our study not only expands the spectrum of COL4A5 mutations, but also analysis the progress of AS and fills the knowledge about course and potential further complications and health risks of AS.

Introduction

Alport syndrome (AS, OMIM: 301050) is characterized by proteinuria, hematuria, progressive glomerulonephritis with a high life-time risk for end-stage renal disease (ESRD), sensorineural hearing loss and ocular abnormalities [1-4]. In addition, several ophthalmic complications may also appear in this disorder. The estimated prevalence of this disorder is more than 0.01%.

Previous study have demonstrated collagen IV α3/α4/α5 network from the basement membranes of the glomerular, lens capsule, cornea and retina are related to AS [5]. Mutations in the COL4A3 (NM_000091), COL4A4 (NM_000092) and COL4A5 (NM_033380) genes may break the stability of collagen IV α3/α4/α5 network and lead to AS [6-8]. However, due to genetic heterogeneity, high cost of testing technique and lacking awareness of testing, there are still a lot of AS patients lacking genetic testing and long-term follow-up records.

In this research, we investigate a family diagnosed as AS. An obvious X-dominant inheritance has been observed in this family. By using whole-exome sequencing, a novel insertion mutation (c.348_349insTCCGG/p.G117SfsX40) of COL4A5 in X chromosome was identified. We also collected and summed up a follow-up clinic data of the proband from 2003 to 2016, this data may help us know more about the progress of this disease.

Subjects and methods

Patients and subjects

This research has been approved by Third Xiangya Hospital of the Central South University. All subjects have consented to this study. In this study, a family with 15 members across four generations from Hunan Province was investigated. The proband and seven family members were diagnosed with AS (Table 1).

Table 1.

Summary of a family with Alport syndrome

Family member	Disease	Sex	Age	COL4A5		Prediction by programs
				DNA	Protein	Polyphen2	SIFT	MutationTaster
III2	Alport syndrome	Male	34 y	c.348_349insTCCGG	p.G117Sfs×40	Probably damaging	Deleterious	Disease causing
I1	Mild hematuria	Female	-	-	-
I2	NO	Male	79 y	-	-
II1	NO	Male	54 y	-	-
II2	Mild hematuria	Female	55 y	c.348_349insTCCGG	p.G117Sfs×40	Probably damaging	Deleterious	Disease causing
II3	Alport syndrome	Male	58 y	c.348_349insTCCGG	p.G117Sfs×40	Probably damaging	Deleterious	Disease causing
II4	NO	Female	60 y	-	-
III1	NO	Female	30 y	-	-
III3	Uremia	Male	-
III4	NO	Male	40 y	-	-
III5	Mild hematuria	Female	39 y	c.348_349insTCCGG	p.G117Sfs×40	Probably damaging	Deleterious	Disease causing
IV1	Mild hematuria	Female	7 y	c.348_349insTCCGG	p.G117Sfs×40	Probably damaging	Deleterious	Ddisease causing
IV2	NO	Male	6 y	-	-
IV3	Alport syndrome	Male	19 y	c.348_349insTCCGG	p.G117Sfs×40	Probably damaging	Deleterious	Disease causing
IV4	NO	Female	15 y	-	-

Whole-exome sequencing

Genomic DNA was extracted with a DNeasy blood and tissue kit (Qiagen, Valencia, Calif., USA) [9]. The Novogene Bioinformatics Institute (Beijing, China) provided the exome capture, high throughput sequencing and common filtering. All the exomes were captured by means of Agilent SureSelect Human All Exon V5 kits and were sequenced with an Illumina HiSeq X Ten platform. Filtering strategies referred to our previous study [10].

Co-segregation analysis

Segregation analysis was applied in all family members according to the whole-exome sequencing results. Primer pairs were designed by Primer 5 and the sequences of primers will be provided upon request.

Results

Clinical features

We described a Chinese family with AS (Table 1; Figure 1A). The proband (III2), a 34-year-old officer from the Central-South China (Changsha, Hunan), had a 13-year history of chronic kidney disease (CKD) and diagnosed as AS. His younger brother (III-3) died from uremia in 2008, the affected male members (II-3, IV-3) were all diagnosed as typical AS and some female members (I-1, II-2, III-5, IV-1) were detected with mild hematuria, no other symptoms were observed in these female members.

Figure 1.

A. Pedigree involved in this study, there were 15 numbers totally. □, male members; ○, female members; Mark 1, deceased male member who was affected by uremia; Mark 2, deceased affected female member with mild hematuria; Mark 3, affected female members with mild hematuria, who has the insertion mutation (c.348_349insTCCGG/G117Sfs*40); Mark 4, the proband; ■, affected male members who was diagnosed as typical Alport syndrome. B. Analysis of COL4A5. The chromatograms show partial sequence of COL4A5, c.348_349insTCCGG/G117Sfs*40, they are control, female patient and male patient. C. Conservation analysis of COL4A5. G117 sites are highlighted in blue. It is highly conserved at protein level across human, Ptroglodytes, Mmusculus, Trubripes, Drerio, Celegans, Xtropicalis, etc.

Genetic analysis

Data filtering excluded shared common variants present in YH database, ESP database, 1000 Genomes Project and dbSNP132. About 1127 unique single nucleotide polymorphisms (SNPs) were detected in this family. After AS-related genes filtering, only a novel insertion mutation (c.348_349insTCCGG/p.G117SfsX40) of COL4A5 in X chromosome was identified in the proband. Sanger sequencing have confirmed that this novel mutation was co-segregated with all the family members. (Figure 1B), which indicates that this insertion mutation in COL4A5 may underlie this inherited disorder. Cross-species alignment analysis of COL4A5 amino acid sequences revealed that this mutated site was highly evolutionarily conserved (Figure 1C).

Long-term follow-up data

All the follow-up data of the proband are shown in Table 2. The proband was first presented at our hospital with proteinuria and hematuria in 2003. In the past decades, he received the drug therapy by Uremic Clearance Granule, Jinshuibao capsule, Bailing Capsule and other ancillary drugs. The follow-up was also started at this time. The cataract and respiratory tract infection was appeared during this time (2003-2007). In 2007, he was diagnosed with AS. During 2007 to 2014, the patient received follow-up visit 12 times and his symptom of the disease became more severe (from CKD3 to CKD5). In 2014, the proband received a kidney transplant but proteinuria symptom appeared again with an unexplained reason in 2016. Therefore, he has visited the hospital for further treatment recently.

Table 2.

Long-term follow-up of the patients with Alport syndrome

Time/phenotype		2003.11	2006.2	2007.6	2007.11	2007.12.	2011.1	2011.7	2011.1	2012.3	2012.7
Kidney	Proteinuria	++++	+++	++	+++	++	++	++	+++	+++	+++
	Hematuria (RBC/mL)	100000	35000	25000	8500	15000	7500	40000	150000	2000	20000
Sensorineural deafness		-	-	-	-	-	-	-	-	-	-
Cataracts		-	-	-	-	√	√	√	√	√	√
Trachea		Pharynx swelling	-	Cough	Sore throat	-	-	-	-	-	Cough
Esophagus		Pharynx swelling	-	-	Sore throat	-	-	-	-	-	-
Anemia		-	-	-	-	-	-	-	-	-	-
Stages		-	-	-	CKD3	CKD3	CKD3	CKD3	CKD3	CKD4	CKD3
Alport syndrome		-	-	-	-	√	√	√	√	√	√
Kidney transplant		-	-	-	-	-	-	-	--		-
Time/phenotype		2012.12	2013.5	2013.1	2013.12	2014.5	2014.6	2014.8	2014.11	2015.4	2016.4
Kidney	Proteinuria	+++	+++	++	++	++	++	-	-	-	++
	Hematuria (RBC/mL)	65000	67500	10000	27500	12500	10000	-	-	<8000	<8000
Sensorineural deafness		-	-	-	-	-	-	-	-	-	-
Cataracts		√	√	√	√	√	√	√	√	√	√
Trachea		-	-	-	-	-	-	-	Cough	-	-
Esophagus		-	-	-	-	-	-	--	-	-	-
Anemia		√	√	-	√	√	√	√	-	-	-
Stages		CKD3	CKD3	CKD3	CKD3	CKD3	CKD4	CKD5	-	-
Alport syndrome		√	√	√	√	√	√	√	√	√	√
Kidney transplant		-	-	-	-	-	-	√	-	-	-

Discussion

In this study, we applied whole-exome sequencing to identify the possible pathogenic genes for the family with AS. A novel insertion mutation (c.348_349insTCCGG/p.G117Sfs×40) in COL4A5 was identified in this family. This insertion mutation locates in exon 6 of COL4A5, a highly conserved collagenous domain with 1430 residues, which leads to a truncated protein. Our genetic study confirmed the clinic diagnosis of AS.

COL4A5 gene contains 51 exons, encoding type IV collagen α5 chain which could combine with COL4A3 and COL4A4, forming triple-helical domains which located in glomerular basement membrane. Type IV collagen α5 chain contains 1,685 amino acid residues, including a 26-residue signal peptide, a 1,430-residue collagenous domain and a 229-residue carboxyl-terminal non-collagenous domain (NC1 domain) [11-13]. The 1,430-residue collagenous domain is the most significant domain, which plays a crucial role in the combination with COL4A3 and COL4A4. But this insertion mutation would interrupt its binding domain and affect triple-helical domains’ function, finally, leading to AS [14].

Because COL4A5 is too long to detect via Sanger sequencing, most of COL4A5 mutations were identified by next-generation sequencing in recent years. COL4A5 is located at Xq22.3 [7,15]. Disease related to this gene shows a gender difference in severity with males showing more severe symptoms. Only 12% female patients with X-linked AS reach end-stage renal disease before the age of 40 and only 30% before the age of 60 in European cohort [16,17]. While 70% male patients with X-linked AS had end-stage renal disease before the age of 30 and the remaining 30% after their 30 [18,19]. In our study, males with AS exhibited more severe symptoms than females, which is consistent with previous studies [16-19].

In this study, we also performed a long-term follow-up of an AS patient with COL4A5 mutation. The clinic data from the first hospitalization with proteinuria and hematuria in 2003 to renal transplant in 2014. We can conclude that this disease was a progressive disease, especially the progress of chronic kidney disease. Our concluded data may contribute to fill the knowledge about course and potential further complications and health risks of AS.

To sum up, we identified a novel insertion mutation (c.348_349insTCCGG/p.G117Sfs×40) of COL4A5 in X chromosome by whole-exome sequencing in a family with AS. And we performed a long-term follow-up for about 15 years of the proband. Our research not only expands the spectrum of COL4A5 mutations, but also provides more knowledge about course and potential further complications and health risks during adulthood and advanced age.

Disclosure of conflict of interest

None.