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Journal of the American Society of Nephrology : JASN logoLink to Journal of the American Society of Nephrology : JASN
. 2013 Sep 19;24(12):1945–1954. doi: 10.1681/ASN.2012100985

COL4A3/COL4A4 Mutations and Features in Individuals with Autosomal Recessive Alport Syndrome

Helen Storey *,✉, Judy Savige †, Vanessa Sivakumar †, Stephen Abbs ‡, Frances A Flinter §
PMCID: PMC3839543  PMID: 24052634

Abstract

Alport syndrome is an inherited disease characterized by hematuria, progressive renal failure, hearing loss, and ocular abnormalities. Autosomal recessive Alport syndrome is suspected in consanguineous families and when female patients develop renal failure. Fifteen percent of patients with Alport syndrome have autosomal recessive inheritance caused by two pathogenic mutations in either COL4A3 or COL4A4. Here, we describe the mutations and clinical features in 40 individuals including 9 children and 21 female individuals (53%) with autosomal recessive inheritance indicated by the detection of two mutations. The median age was 31 years (range, 6–54 years). The median age at end stage renal failure was 22.5 years (range, 10–38 years), but renal function was normal in nine adults (29%). Hearing loss and ocular abnormalities were common (23 of 35 patients [66%] and 10 of 18 patients [56%], respectively). Twenty mutation pairs (50%) affected COL4A3 and 20 pairs affected COL4A4. Of the 68 variants identified, 39 were novel, 12 were homozygous changes, and 9 were present in multiple individuals, including c.2906C>G (p.(Ser969*)) in COL4A4, which was found in 23% of the patients. Thirty-six variants (53%) resulted directly or indirectly in a stop codon, and all 17 individuals with early onset renal failure had at least one such mutation, whereas these mutations were less common in patients with normal renal function or late-onset renal failure. In conclusion, patient phenotypes may vary depending on the underlying mutations, and genetic testing should be considered for the routine diagnosis of autosomal recessive Alport syndrome.

Alport syndrome is an inherited renal disease characterized by hematuria, progressive renal failure, hearing loss, and ocular abnormalities. Alport commented in 1927 that the occurrence of hematuria and hearing loss in a pedigree was not coincidental but represented a clinical syndrome, and that the more severe disease in male individuals was consistent with X-linked inheritance.1 We now understand that nearly 85% of patients have X-linked disease due to a pathogenic mutation in the COL4A5 gene, and the remaining individuals usually have autosomal recessive inheritance with two pathogenic mutations in either the COL4A3 or COL4A4 gene.

Alport syndrome is usually suspected when the typical clinical features are present. Diagnostic features2 include a positive family history, a lamellated glomerular basement membrane (GBM),3 high tone sensorineural hearing loss, and lenticonus and macular flecks on ophthalmoscopy.4 However, these features do not distinguish between X-linked and autosomal inheritance. The possibility of autosomal recessive disease is often overlooked, but its recognition is important because the genetic implications are different for the patient and other family members. Affected male individuals with X-linked disease, but few female individuals, eventually develop renal failure and the disease is transmitted from one generation to another. With autosomal recessive inheritance, male and female individuals are equally likely to be affected; renal failure tends to occur in only one generation except in the presence of multiple consanguinity. In our previous report of 206 patients referred for molecular testing of COL4A5, the pathogenic mutation detection rates in families fulfilling none, one, two, three, or four diagnostic criteria were 0%, 18%, 64%, 89%, and 81%, respectively. Autosomal recessive inheritance was suspected to account for the families meeting four diagnostic criteria in whom no pathogenic COL4A5 mutation was detected.5

Nearly 300 pathogenic mutations have been described in the COL4A3 and COL4A4 genes (Leiden Open Variation Database; https://grenada.lumc.nl/LOVD2/COL4A/home.php?action=switch_db), but many of these are from patients with thin basement membrane nephropathy (TBMN). There are few reports describing two pathogenic mutations in individuals with autosomal recessive Alport syndrome.6–16 Even fewer studies have examined how mutations may determine clinical features.

Here we describe genetic mutations and clinical features in 40 patients in whom two pathogenic mutations were identified in the COL4A3 or COL4A4 gene, consistent with the diagnosis of autosomal recessive Alport syndrome. In many cases, the mutations were demonstrated to be in trans, which is on different chromosomes, confirming autosomal recessive inheritance. Testing examined the entire coding region and splice sites of both COL4A3 and COL4A4 using unidirectional fluorescent Sanger DNA sequencing, analyzed using Mutation Surveyor software. For detecting point mutations in the regions screened, this approach has an analytical sensitivity and specificity of >99%.17

Results

Clinical Features

Between August 2009 and December 2011, 205 apparently unrelated British and Australian patients with clinically suspected autosomal recessive Alport syndrome or TBMN were referred for genetic testing.

Ninety-four individuals (46%) were found to have at least one pathogenic mutation and an additional 36 individuals (17%) had at least one variant of unknown significance. Forty participants (20%) had two pathogenic mutations in COL4A3 or COL4A4 (Table 1); in 18 families, these mutations were confirmed to be present in trans in the index cases. In 7 of 12 families, dosage analysis for the corresponding exon confirmed the presence of two alleles in individuals apparently homozygous for mutations.

Table 1.

Pathogenic COL4A3 and COL4A4 mutations detected in 40 patients with autosomal recessive Alport syndrome

Patient No. H/h COL4A3/COL4A4 Mutation 1 Exon Novel? Mutation 2 Exon Novel? Sex Ethnicity Age (yr)a Age at Renal Failure (yr) Renal Biopsy Hearing Loss Characteristic Eye Signs Notes
1 h COL4A3 c.162dup 3 Yes c.2313_2330del 30 Yes M Tasmanian family, British origin generations ago 34 23 Yes Yes Yes Mutations in trans
p.(Gly55fs)b p.(Leu775_Gly780del)
2 h COL4A3 c.391G>T 7 Yes c.2621_2622delinsT 32 No, AR AS, H, h8 F Australian family probably originally from UK 38 15 Yes Yes Yes
p.(Glu131*)b p.(Gly874fs)b
3 h COL4A3 c.461G>C 8 Yes c.794G>A 14 Yes M English 44 37 Yes No No Mutations in trans
p.(Gly154Ala) p.(Gly265Glu)
4 h COL4A3 c.546G>T 9 Yes c.4981C>T 52 No, AR AS, h8 F Australian family, probably originally from UK 38 N/A Yes Yes No c.546G>T has splice effect
p.(Gln182His) p.(Arg1661Cys)
5 h COL4A3 c.663_664del 12 Yes c.1937dup 27 Yes F English 10 N/A (N @ 9) Yes No NK Mutations in trans
p.(Arg221fs)b p.(Glu647fs)b
6 h COL4A3 c.713del 13 Yes c.1918G>A 26 No, AR AS8 F NK 21 N/A (N @ 17) N @ 6 No No
p.(Pro238fs)b p.(Gly640Arg)
7 h COL4A3 c.1085del 19 Yes c.3580del 42 Yes M NK 6 N/A (N @ 6) Yes No NK Mutations in trans
p.(Pro362fs)b p.(Arg1194fs)b
8 H COL4A3 c.1409–5T>A 22i No, AR AS18 c.1409–5T>A 22i No, AR AS18 M Australian, parents were Italian 54 38 Yes Yes Yes Apparent homozygous. Parents first cousins
9 h COL4A3 c.2031_2038dup 28 Yes c.3472G>C 40 Yes M NK Deceased 26 Yes Yes No
p.(Gly680fs)b p.(Gly1158Arg)
10 h COL4A3 c.2031_2038dup 28 Yes c.3210+1G>A 37i Yes M NK 41 10 Yes Yes Yes
p.(Gly680fs)b
11 h COL4A3 c.2083G>A 28 No, TBMN27 c.2452G>A 31 Yes M English 19 N/A (N @ 18) Yes No NK Mutations in trans
p.(Gly695Arg) p.(Gly818Arg)
12 H COL4A3 c.2567G>A 32 Yes c.2567G>A 32 Yes M NK 16 N/A (N @ 16) Yes Yes No Apparent homozygous; father heterozygous
p.(Gly856Glu) p.(Gly856Glu)
13 h COL4A3 c.2621_2622delinsT 32 No, AR AS, H and h8 c.3866del 43 Yes F NK 40 17 Yes Yes Yes
p.(Gly874fs)b p.(Gly1289fs)b
14 h COL4A3 c.2745_2746+7del 33–33i Yes c.4981C>T 52 No, AR AS, h8 F NK 14 NK Yes No No Mutations in trans
p.(Gly916fs)b p.(Arg1661Cys)
15 H COL4A3 c.2768_2778del 34 Yes c.2768_2778del 34 Yes F Australian family, probably originally from UK 44 25 Yes Yes Yes Confirmed homozygous; consanguineous
p.(Val923fs)b p.(Val923fs)b
16 h COL4A3 c.2768_2778del 34 Yes c.4981C>T 52 No, AR AS, h8 F NK 49 NK NK NK NK Mutations in trans
p.(Val923fs)b,c p.(Arg1661Cys) +
17 h COL4A3 c.2768_2778del 34 Yes c.3760G>C 43 Yes F Mixed British 36 23 Yes Yes Yes
p.(Val923fs)b,c p.(Gly1254Arg) +
18 h COL4A3 c.3472G>C 40 Yes c.4994G>A 52 Yes F English 17 N/A (N @ 17) TBM@5 No NK Mother has one mutation
p.(Gly1158Arg) p.(Cys1665Tyr)
19 h COL4A3 c.4030del 46 Yes c.4408G>T 48 Yes M Punjabi 8 N/A (N @ 7) Yes No NK Mother has one mutation
p.(Val1344fs)b p.(Gly1470Trp)
20 H COL4A3 c.4347_4353del 48 Yes c.4347_4353del 48 Yes M NK 31 17 Yes Yes NK Apparent homozygous. Nonconsanguineous
p.(Arg1450fs)b p.(Arg1450fs)b
21 h COL4A4 c.81_86del 3 Yes c.2906C>G 32 No, AR AS, TBMN9,11,18,19 F NK 15 NK Yes NK NK Father has one mutation
p.(Ile29_Leu30del) p.(Ser969*)b
22 h COL4A4 c.81_86del 3 Yes c.2906C>G 32 No, AR AS, TBMN9,11,18,19 M NK 10 NK Yes NK NK
p.(Ile29_Leu30del) p.(Ser969*)b
23 h COL4A4 c.1099+1G>A 18i Yes c.2638del 30 No, AR AS9 M Tasmanian family, likely originally from UK 30 23 Yes Yes Yes
p.(Ala880fs)b
24 H COL4A4 c.1598G>A 22 Yes c.1598G>A 22 Yes F NK 20 N/A (N @ 20) Yes No NK Confirmed homozygous
p.(Gly533Asp) p.(Gly533Asp)
25 H COL4A4 c.1598G>A 22 Yes c.1598G>A 22 Yes F Slovakian 30 N/A (N @ 30) NK Yes NK Confirmed homozygous Nonconsanguineous
p.(Gly533Asp) p.(Gly533Asp)
26 H COL4A4 c.1802del 24 No, AR AS7 c.1802del 24 No, AR AS7 F NK 20 N/A (N @ 20) Yes No No Apparent homozygous; some consanguinity
p.(Pro601fs)b p.(Pro601fs)b
27 h COL4A4 c.2590G>A 30 No, AR AS15 c.2906C>G 32 No, AR AS, TBMN9,11,18,19 F NK 42 N/A Yes No No Mutations in trans
p.(Gly864Arg) p.(Ser969*)b
28 h COL4A4 c.2638del 30 No, AR AS9 c.2884G>T 32 Yes M Australian 32 28 NK NK NK
p.(Ala880fs)b p.(Glu962*)b
29 H COL4A4 c.2638del 30 No, AR AS9 c.2638del 30 No, AR AS9 M NK 20 NK Yes Yes NK Confirmed homozygous
p.(Ala880fs)b p.(Ala880fs)b
30 h COL4A4 c.2744del 31 No, AR AS9 c.3197G>T 34 Yes F Australian family, likely originally from UK 28 35 NK NK NK
p.(Gly915fs)b p.(Gly1066Val)
31 h COL4A4 c.2906C>G 32 No, AR AS, TBMN9,11,18,19 c.3215G>T 35 Yes F NK 23 17 Yes Yes NK Mutations in trans
p.(Ser969*)b p.(Gly1072Val)
32 H COL4A4 c.2906C>G 32 No, AR AS, TBMN9,11,18,19 c.2906C>G 32 No, AR AS, TBMN9,11,18,19 M Australian, parents were British 34 28 Yes Yes Yes Confirmed homozygous Parents first cousins
p.(Ser969*)b p.(Ser969*)b
33 h COL4A4 c.2906C>G 32 No, AR AS, TBMN9,11,18,19 c.4538G>A 47 Yes M NK 39 22 Yes Yes NK
p.(Ser969*)b p.(Cys1513Tyr)
34 h COL4A4 c.2906C>G 32 No, AR AS, TBMN9,11,18,19 c.4781_4807dup 47 Yes F NK 41 N/A (N @41) Yes Yes NK Mutations in trans
p.(Ser969*)b p.(Ser1594_Leu1602dup)
35 H COL4A4 c.2906C>G 32 No, AR AS, TBMN9,11,18,19 c.2906C>G 32 No, AR AS, TBMN9,11,18,19 M NK 36 18 Yes Yes NK Confirmed homozygous
p.(Ser969*)b p.(Ser969*)b
36 H COL4A4 c.2906C>G 32 No, AR AS, TBMN9,11,18,19 c.2906C>G 32 No, AR AS, TBMN9,11,18,19> F NK 35 NK Yes Yes NK Apparent homozygous
p.(Ser969*)b p.(Ser969*)b
37 h COL4A4 c.3602G>A 39 Yes c.4376G>T 46 Yes F English 32 N/A (N @ 30) Yes No NK Sister has one mutation
p.(Gly1201Asp) p.(Gly1459Val)
38 h COL4A4 c.4200_4201del 44 Yes c.4522G>A 46 Yes M Scottish 46 21 Yes Yes Yes Mutations in trans
p.(Gly1401fs)b p.(Gly1508Ser)
39 h COL4A4 c.4444dup 46 Yes c.4763G>A 47 Yes M English 29 20 Yes Yes NK Sister has one mutation
p.(Leu1482fs)b p.(Cys1588Tyr)
40 H COL4A4 c.4788G>A
 47 Yes c.4788G>A 47 Yes F NK 17 12 Yes Yes NK Confirmed homozygous– parents are cousins
p.(Trp1596*)b p.(Trp1596*)b
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Sequence nomenclature is based on COL4A3 reference sequence LRG_230 (NM_000091.4) and COL4A4 reference sequence LRG_231 (NM_000092.4), in which nucleotide number 1 corresponds to the first base of the translation initiation codon. AR AS, autosomal recessive Alport syndrome; I, intron; F, female; H, homozygous; h, heterozygous; M, male; N, normal; N/A, not appropriate; NK, not known.

a

Age when clinically assessed.

b

A pathogenic mutation resulting in a stop codon (i.e., a nonsense or frameshift mutation).

c

The mutation was originally identified in Germany (Center for Nephrology and Metabolic Disorders, Germany).

The 40 patients with autosomal recessive Alport syndrome comprised 31 British individuals and 9 Australians, 7 of whom were of British origin. None of the participants was known to be related at the time of testing; however, at a subsequent clinic visit, members of two families recognized each other and the index cases were confirmed to be second cousins (patients 23 and 28).

This cohort included 19 male participants (48%) and 21 female participants (52%). Their median age at the time of study was 31 years (range, 6–54), and nine participants (23%) were aged ≤18 years.

Five of 9 children (56%) and 9 of 31 adults (29%; median age 25.5 years; range, 19–42) had normal renal function, and 20 of 34 patients (59%) had end stage renal failure. The median age at onset of renal failure was 22.5 years (range, 10–38). Within individual families, the age at which affected individuals reached end stage renal failure varied by up to 9 years.

Thirty-four of 36 patients (94%) had a lamellated GBM on renal biopsy. However, two adults who had a biopsy as children (aged 5 and 6 years) had a normal or thinned GBM, respectively. Four patients had no renal biopsy or record of the GBM appearance.

Extrarenal features were common. Twenty-three of 35 individuals (66%) had hearing loss, and 10 of 18 individuals (56%) had lenticonus or retinopathy.

Pathogenic Mutations

Eighty pathogenic mutations were identified (Table 1). We found 68 different variants, with 12 homozygous changes, and 9 variants were present in multiple individuals. Twenty patients (50%) had two pathogenic mutations in the COL4A3 gene and 20 patients (50%) had mutations in the COL4A4 gene, while 39 variants were novel (Table 2).

Table 2.

Summary of pathogenic COL4A3 and COL4A4 mutations found in 40 autosomal recessive Alport syndrome patients

Patient/Mutation Features COL4A3 Gene COL4A4 Gene Total for All Index Cases
Patients (% of all patients)
 With mutation affecting gene 20 (50) 20 (50) 40 (100)
 With homozygous mutations 4 (10) 8 (20) 12 (30)
 With compound heterozygous mutations 16 (40) 12 (30) 28 (70)
Total mutationsa 36 32 68
  Previously reported 8 15 23
 Novel 28 17 45
Unique mutations 29 20 49
 Previously reported 5 5 10
 Novel 24 15 39
Mutation type (% of mutations found in that gene)a
 Frameshift 17 (47) 7 (22) 24 (35)
 Nonsense 1 (3) 11 (35) 12 (18)
 Missense 15 (41) 10 (31) 25 (37)
[10 involving Gly (28)] [8 involving Gly (25)]
[4 involving Cys (11)] [2 involving Cys (6)]
 Small deletion/duplication 1 (3) 3 (9) 4 (6)
 Splicing 2 (6) 1 (3) 3 (4)
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a

Homozygous mutations only counted once.

Five individuals were from known consanguineous families (patients 8, 15, 26, 32, and 40) and an additional 7 families were also demonstrated to have homozygous mutations, making a total of 12 families (30%). These included four homozygous COL4A3 mutations and eight homozygous COL4A4 mutations. The remaining 28 individuals (70%) had compound heterozygous COL4A3 or COL4A4 mutations.

Of the nine variants found on several occasions, five were COL4A3 mutations and four were COL4A4 mutations. The COL4A3 mutations included c.2031_2038dup (p.(Gly680fs)) in two individuals, c.2621_2622delinsT (p.(Gly874fs)) in two individuals,8 c.2768_2778del (p.(Val923fs)) occurring four times in three individuals, c.3472G>C (p.(Gly1158Arg)) in two individuals, and c.4981C>G (p.(Arg1661Cys)) in three individuals.8

The COL4A4 mutations included c.81_86del (p.(Ile29_Leu30del)) in two individuals, c.1598G>A (p.(Gly533Asp)) four times in two individuals, c.2638del (p.(Ala880fs)) four times in three individuals,9 and c.2906C>G (p.(Ser969*)) 12 times in nine individuals (23% of our patients). The c.2906C>G (p.(Ser969*)) variant in COL4A4 was the most common mutation detected in this series, and was described previously in both autosomal recessive Alport syndrome and TBMN.9,11,18,19

Of the 68 pathogenic mutations detected, 25 (37%) resulted in a missense change, 24 (35%) were frameshift mutations, 12 (18%) were nonsense mutations, 4 (6%) were small deletions/duplications, and 3 (4%) were splicing mutations (Table 1). Of the 24 frameshift mutations, 17 affected COL4A3 and 7 affected COL4A4; of the 12 nonsense mutations, 1 affected COL4A3 and 11 were found in COL4A4, 9 of which were the same c.2906C>G change (Table 2). Of the 25 missense mutations, 15 affected COL4A3 and 10 were found in COL4A4.

The most common missense mutations affected a glycine residue (18 of 25, 72%) and the most common substitution was with arginine (7 of 18, 39%). Interestingly, six of the remaining missense mutations abolished (one mutation in COL4A3 and two in COL4A4) or created (p.(Arg1661Cys) in COL4A3) a cysteine residue in the noncollagenous domains.

There were 31 patients (78%) with at least one (n=18, 45%) or two (n =13, 33%) pathogenic mutations that resulted directly, or indirectly through a frameshift, in a stop codon. All 17 individuals who developed end stage renal failure before age 30 years had at least one mutation resulting in a stop codon, with 8 individuals having two mutations. Of the nine adults with normal renal function, three had one mutation resulting in a stop codon (normal renal function at ages 17, 41, and 42 years), and one patient had two (normal renal function at age 20 years). Two of the three patients who developed late-onset renal failure (after 30 years) had no mutations resulting in a stop codon.

The two patients in the study who were second-degree cousins (patients 23 and 28) shared a pathogenic mutation in COL4A4 [c.2638del; p.(Ala880fs)], but each had a different second pathogenic mutation in COL4A4 [c.1099+1G>A and c.2884G>T p.(Glu962*), respectively]. The second mutations were not found in any other individuals in this study, and had not been described before. In addition, the families of two unrelated male participants (patients 22 and 38) each comprised three affected brothers, all with autosomal recessive Alport syndrome. Both parents of another patient (patient 17) were each carriers of one of the pathogenic mutations; however, the 74-year-old father had hematuria and anterior lenticonus but normal renal function, possibly due to somatic mosaicism involving the COL4A3 c.3760G>C (p.(Gly1254Arg)) mutation. (To note, somatic mosaicism is the coexistence of genetically distinct cell populations in a single individual, usually as a result of postzygotic genetic events that can occur at any time in the life cycle. A person who is mosaic for a somatic mutation may or may not be affected by the disorder caused by that mutation. Individuals will express the phenotype depending on how many and which cells are affected. Typically, individuals with somatic mosaicism exhibit a milder phenotype because only a proportion of cells contain the mutation and/or because the mutation is confined to a finite segment of the body.)

Discussion

Improved mutation detection techniques for autosomal recessive Alport syndrome have increased the sensitivity of genetic testing. This study demonstrated the pathogenic variants in 40 apparently unrelated individuals with autosomal recessive Alport syndrome. This series represents the largest single cohort of patients with autosomal recessive Alport syndrome in which both pathogenic mutations were identified. It also correlates the genotype with clinical features. Previously, data were published on only 40 patients in whom two pathogenic mutations have been identified.

Clinical data were obtained from the referral forms accompanying samples for testing and were sometimes incomplete. Nevertheless, this study clearly demonstrates the onset of end stage renal failure in autosomal recessive Alport syndrome in both children and in adults even into middle age. Interestingly, renal function remained normal in some adults as well as children. This represents the first description of mild renal disease in autosomal recessive Alport syndrome. Where renal failure occurred, it was more common with a nonsense mutation or a mutation that resulted in a downstream stop codon. Renal failure, hearing loss, and ocular abnormalities were common in both male and female participants. The age at onset of renal failure across the cohort varied more than seen in X-linked Alport syndrome. Most patients had a lamellated GBM on renal biopsy, but one child had thinning only and another had a normal GBM. These changes resemble those seen in X-linked disease, in which minor, sometimes nonspecific, histologic abnormalities are common in early disease.

It was not possible to determine the general mutation detection rate or the numbers of patients referred for specific types of disease, because referral information was often nonspecific; it was unclear how many patients were referred with suspected autosomal recessive Alport syndrome rather than TBMN, autosomal dominant Alport syndrome, or another inherited renal disease. In addition, some patients who may have had autosomal recessive Alport syndrome would not have been included in this analysis because only one pathogenic mutation was detected.

In this study, direct DNA sequencing was used to detect pathogenic mutations, but this technique still overlooks exonic deletions/duplications as well as deep intronic variants. Large deletions and duplications account for approximately 12% of cases of X-linked Alport syndrome, and deep intronic variants in COL4A5 have also been demonstrated.20 Although the mutation spectrums of the genes do differ, it seems possible that large deletions/duplications and intronic variants in COL4A3 and COL4A4 could account for a number of mutations currently not being detected. However, some patients in which only one mutation was detected may suffer from TBMN, the prognosis of which can be worse than originally thought,21 rather than Alport syndrome, in which case only one pathogenic mutation would be expected to be detected. The distinction between Alport syndrome and TBMN may be difficult to determine but is critical.22

Immunohistochemical analysis of basement membrane type IV collagen expression, with a skin or renal biopsy, has also been reported as a useful diagnostic tool; however, there is a significant false negative rate,23,24 and renal biopsies are an invasive procedure. In approximately 80% of male individuals with X-linked Alport syndrome, GBM staining for α3(IV), α4(IV), and α5(IV) is completely negative.25 Although a skin biopsy can be a cost-effective and simple diagnostic test, discordance of expression between the epidermal basement membrane and GBM has been reported.26 Therefore, immunohistochemistry is not always reliable and is not widely offered in the United Kingdom.

Most mutations in this cohort were novel and there were no “hot spots” (regions of DNA where mutations are observed with greater frequency) in COL4A3 or COL4A4; however, some mutations recurred in different patients. The high proportion of new mutations suggested that many more are still to be identified. Ten mutations that had been described previously in Alport syndrome or TBMN were present in 22 of the patients (Leiden Open Variation Database, https://grenada.lumc.nl/LOVD2/COL4A/home.php?action=switch_db).7–9,11,15,18,19,27

Nine mutations were found more than once. The most common variant, p.(Ser969*), was detected 12 times in nine individuals (23% of patients) and represented 13% (9 of 68) of all mutations in this cohort. This variant was found not only in the British patients but also in the Australians of British origin. This variant has not been reported in other populations, such as the European or Cypriot communities, but appears due to a “founder” effect.9 Founder mutations have been described in the COL4A5 gene in North America in X-linked Alport syndrome, and other variants have also been reported in the COL4A3 and COL4A4 genes in Cyprus.28–30

Only five individuals were known to be from consanguineous families but 12 had homozygous mutations. Two of these mutations, including p.(Ser969*), were found in multiple individuals and the homozygosity likely resulted from the high background prevalence of this variant rather than recent consanguinity.

Nonsense mutations or mutations resulting in downstream stop codons were the most common variants, occurring in 78% of patients (31 of 40). These types of mutations were associated with early onset renal failure. Nonsense mutations result in the loss of collagen IV α3α4α5 heterotrimer from the GBM,31 whereas missense mutations produce a structurally abnormal GBM that is less likely to be deleterious. This study is the first that indicates that the nature of the underlying mutations in autosomal recessive Alport syndrome may also affect the clinical phenotype.

Most missense mutations in this study affected glycine residues and the most common change resulted in an arginine substitution. The p.(Arg1661Cys) mutation in COL4A3 was previously reported in Alport syndrome8 and involves an exposed arginine residue that is conserved in all six collagen IV chains. This mutation likely results in mismatched disulphide bond formation, perhaps altering the conformation of the NC1 domain or leading to protomer instability.8,32 This is likely to be due to a founder effect.8 Interestingly, several of the missense mutations described here either abolished or created a new cysteine residue. Cysteines in the NC1 domains of collagen IV are highly conserved between and within species and are important for intramolecular disulphide bond formation.33,34 In addition, the NC1 domain plays an important role in heterodimerization during collagen chain formation and assembly.35–37

These genotype-phenotype correlations in autosomal recessive Alport syndrome are similar to those observed in X-linked Alport syndrome, in which mutations that directly or indirectly result in nonsense mutations are associated with early onset renal failure compared with missense mutations that result in less severe disease.25,38

When an individual is diagnosed with Alport syndrome, it is important to distinguish between X-linked and autosomal recessive inheritance because of the different risks of renal failure in other family members. Autosomal recessive inheritance is suspected when disease occurs in a consanguineous family, or a single generation, and where male and female individuals are affected with equal frequency and severity. Recessive inheritance may be suspected where a female individual develops renal failure, especially at a young age, because the minority of female individuals with X-linked Alport syndrome who develop renal failure generally do so when they are older (typically in middle age or later). Examination of the pedigree is not always helpful in distinguishing between the likelihood of X-linked and autosomal recessive disease, and may be misleading. This study did not examine the pedigrees of all affected individuals but did identify two families, initially thought to be X-linked, in which all three sons were affected and another family in which the sons of two female cousins were affected and shared only a single pathogenic variant.

Molecular testing confirms the clinically suspected diagnosis of Alport syndrome and is increasingly available. It is appropriate to consider molecular testing instead of, or before, a renal biopsy because the results are conclusive, indicate the mode of inheritance, and avoid the risks associated with an invasive procedure.39 Once the underlying molecular basis of Alport syndrome is known, the index case and at-risk family members should be offered genetic counseling. Parents of an individual with autosomal recessive Alport syndrome are “obligate carriers” because the new mutation rate is so low. They typically have TBMN and hematuria. Siblings of the index case have a 25% chance of recessive disease too, and carrier couples may wish to consider a range of reproductive options including prenatal and preimplantation genetic diagnosis. Testing can also be used to exclude Alport syndrome in some family members, which allows them to be considered as potential renal donors.

Most apparently sporadic cases of Alport syndrome should first be screened for a pathogenic mutation in COL4A5 but autosomal recessive inheritance must be considered if no pathogenic variant is identified. Where autosomal recessive inheritance is strongly suspected, it is appropriate to test for pathogenic mutations in COL4A3 and COL4A4 in the first instance.

Concise Methods

Study Population

Individuals suspected of having Alport syndrome on clinical features or GBM appearance had been referred by their renal physician or clinical geneticist for genetic testing. Recessive inheritance was suspected in young female participants with renal failure and hearing loss, in the presence of consanguinity, or when disease was observed in only a single generation. Most participants had previously been tested for mutations in COL4A5, with no pathogenic mutation detected.

Genetic testing for pathogenic mutations in COL4A3 and COL4A4 was undertaken at a UK accredited reference laboratory. Patients with two pathogenic mutations identified in the COL4A3 or COL4A4 genes over a 2-year period (August 2009 to December 2011) were identified and clinical data were collected from the patients’ medical records.

Genetic Testing for COL4A3 and COL4A4 Pathogenic Mutations

Genomic DNA was extracted from peripheral blood leukocytes using a Chemagic Magnetic Separation Module I and the 3 ml Chemagic DNA Blood Kit Special (PerkinElmer chemagen Technologie GmbH), and the entire coding regions and splice sites of the COL4A3 (52 exons) and COL4A4 genes (47 coding exons), sequenced using high-throughput unidirectional tagged primer Sanger sequencing, in a 384-well format, and robotics including the Biomek NX Laboratory Automation Workstation (Beckman Coulter), Multiprobe II 8 Tip Robotic Liquid Handling System (PerkinElmer), and Innovadyne Nanodrop II (IDEX Health and Science). PCR products were generated using Qiagen Multiplex PCR Buffer at an annealing temperature of 56°C, and cleaned using AMPure magnetic beads (Agencourt Bioscience Corporation). Sequencing used a BigDye Terminator v3.1 cycle kit (Applied Biosystems) and products were cleaned with CleanSEQ magnetic beads (Agencourt Bioscience Corporation). The sequence was read with an ABI Prism 3730 DNA analyzer (Applied Biosystems) and analyzed using Mutation Surveyor software (SoftGenetics). Any nonpolymorphic variants were confirmed using a fresh DNA dilution by bidirectional tagged primer Sanger DNA sequencing. Screening of DNA for whole gene or exon deletions and duplications was not carried out. Where possible, relatives of the index cases were examined for a heterozygous copy of the mutation to confirm that both mutations were present in trans.

Dosage Analyses

PCR products were generated with unlabeled, tagged gene-specific primers and fluorescently labeled tag primers in Qiagen Multiplex PCR Buffer at an annealing temperature of 56°C. Products were detected using an ABI Prism 3730 DNA analyzer (Applied Biosystems), and the fragments were examined with GeneMarker software (SoftGenetics). The dosage quotient analysis calculation was performed with Microsoft Excel spreadsheet software.

Variant Nomenclature and Classification

Variants were described according to the COL4A3 reference sequence LRG_230 (NM_000091.4) and COL4A4 reference sequence LRG_231 (NM_000092.4), where nucleotide number 1 corresponds to the first base of the translation initiation codon, and using the nomenclature recommended by the Human Genome Variation Society.40

Variant pathogenicity was assessed using Alamut software (versions 1.5 and 2.0; Interactive Biosoftware), which includes tools AlignGVGD, SIFT, and Polyphen, interrogation of dbSNP and the Swissprot databases, interspecies conservation, variant domain location and Grantham distance due to the effect of the mutation, a Google web search, and splicing algorithms SpliceSiteFinder-like, MaxEntScan, NNSplice, and GeneSplicer. Pathogenicity of variants was determined in accordance with the Clinical Molecular Genetics Society best practice guidelines.41

Laboratory and online disease databases were also examined to determine whether the variant had been reported previously and family members were tested where possible to determine whether the variant segregated with disease within the family. All variants were recorded in the COL4A3 and COL4A4 Leiden Open Variation Databases (http://www.lovd.nl/COL4A3 and http://www.lovd.nl/COL4A4).42,43

Disclosures

None.

Acknowledgments

We thank all of the nephrologists and clinical geneticists who have sent samples of blood to our laboratory for molecular testing and provided clinical information about their patients. We also thank our many patients for their interest and support.

This work was funded in part by the Alport Foundation of Australia.

Nine of these patients were described separately in poster form at the American Society of Nephrology Annual Meeting, October 30–November 4, 2012, in San Diego, California, as well as in a separate manuscript.

Footnotes

Published online ahead of print. Publication date available at www.jasn.org.

See related editorial, “The Interface of Genetics with Pathology in Alport Nephritis,” on pages 1925–1927.

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