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Novel COL4A3 Mutations in African American Siblings With AutosomalRecessive Alport Syndrome

Christine Cook, MD,1 Christopher A. Friedrich, MD, PhD,2 and Radhakrishna Baliga, MD1

We describe a novel mutational study in 2 African American siblings with autosomal recessive Alportsyndrome. Both siblings were compound heterozygotes for 2 abnormal DNA sequences in exon 49 ofthe COL4A3 gene, p.Arg1496X (CGA¡TGA) and p.Arg1516X (CGA¡TGA). These are nonsensemutations in the noncollagenous domain resulting in premature termination codons and have not beenpreviously reported. In an African American population in which autosomal recessive Alport syndrome israrely seen, complete sequencing of the COL4A3 and COL4A4 genes may be necessary to identify theunderlying mutation and confirm the diagnosis.Am J Kidney Dis 51:e25-e28. © 2008 by the National Kidney Foundation, Inc.

INDEX WORDS: Alport syndrome; kidney failure; mutation detection; kidney transplant.

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lport syndrome is a progressive hereditarynephropathy often associated with extrare-

al manifestations, including sensorineural deaf-ess and/or ocular abnormalities.1 It is a geneti-ally heterogeneous disease caused by mutationsn type IV collagen of the basement membrane.nheritance patterns include X-linked dominant80%), autosomal recessive (15%), and autoso-al dominant (5%).2 The autosomal recessive

orm is caused by mutations in either the COL4A3r COL4A4 collagen genes. It rarely was re-orted in African Americans. We describe novelonsense mutations in the COL4A3 gene in Afri-an American siblings who are compound het-rozygotes for autosomal recessive Alport syn-rome.

CASE REPORTS

atient 1

A 14-year-old African American girl presented with a-day history of headaches. Her review of systems wasegative except for a 1-year history of progressive hearingoss. On examination, blood pressure was 168/119 mm Hg,ith normal fundi and 1� pitting edema of the lower

xtremities. Serum creatinine level was 4.3 mg/dL (380ol/L), and blood urea nitrogen level was 30 mg/dL (10.7mol/L). Estimated creatinine clearance was 21 mL/min/

.73 m2 (0.35 mL/s). On urinalysis, there was 3� proteinnd moderate blood. Urinary protein-creatinine ratio was 4.0normal, �0.2). Twenty-four–hour urine protein excretionas 5.8 g. Serum total protein level was 7.3 g/dL (73 g/L),

nd albumin level was 3.9 g/dL (39 g/L). Serum cholesterolevel was increased at 251 mg/dL (6.49 mmol/L).

A renal biopsy specimen showed changes consistent withlport syndrome. Audiometry confirmed the presence ofoderate bilateral sensorineural hearing loss. A skin biopsy

pecimen showed uninterrupted staining of epidermal base-

ent membrane for the �5 chain of type IV collagen (CE

merican Journal of Kidney Diseases, Vol 51, No 5 (May), 2008: e

ashtan, Minneapolis, MN). The patient was started onalcium channel blocker therapy. Peritoneal dialysis therapyas initiated 5 months later. One year after her initialresentation, she received a living related donor kidneyransplant from her father. Twenty-seven months after trans-lantation, both the donor and recipient have had no protein-ria or hematuria. Renal function of the recipient remainstable with a serum creatinine level of 1.4 mg/dL (124ol/L), blood urea nitrogen level of 15 mg/dL (5 mmol/L),

nd urinary protein-creatinine ratio of 0.1.

atient 2

The 11-year-old sister of patient 1 was evaluated forematuria and a 2-month history of intermittent headaches.he had no known hearing deficit. Blood pressure was33/80 mm Hg, and physical examination findings werenremarkable. Serum creatinine level was 0.7 mg/dL (62ol/L), and blood urea nitrogen level was 7 mg/dL (2.5mol/L). Estimated creatinine clearance was 124 mL/min/

.73 m2 (2.07 mL/s). On urinalysis, there was 3� proteinnd large blood. Twenty-four–hour urine protein excretionas 2.5 g. Total serum protein level was 5.0 g/dL (50 g/L),

nd albumin level was 3.4 g/dL (34 g/L). Serum cholesterolevel was increased at 253 mg/dL (6.54 mmol/L). The renaliopsy specimen showed findings consistent with Alportyndrome. Audiometry showed bilateral sensorineural hear-ng loss. Treatment with an angiotensin-converting enzyme

From the 1Department of Pediatrics and 2Department ofreventive Medicine, Division of Medical Genetics, Univer-ity of Mississippi Medical Center, Jackson, MS.

Received and accepted in revised form January 31, 2008.riginally published online as doi:10.1053/j.ajkd.2007.09.028n April 2, 2008.

Address correspondence to Radhakrishna Baliga, MD,epartment of Pediatrics, 2500 North State St, Jackson, MS9216. E-mail: rbaliga@ped.umsmed.edu© 2008 by the National Kidney Foundation, Inc.0272-6386/08/5105-0001$34.00/0

doi:10.1053/j.ajkd.2007.09.028

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nhibitor was initiated. One year after the initial diagnosis,erum creatinine level was 1.1 mg/dL (97 mol/L), andstimated creatinine clearance was 89 mL/min/1.73 m2 (1.48L/s).To confirm the diagnosis, �3(IV) and �4(IV) collagen

enes were subjected to direct DNA sequencing (Matoagel, Weißwasser, Germany). Genomic DNA sequencingf the COL4A3 gene was performed by using dye termina-ors, and fragments were separated by using capillary electro-horesis on an ABI Prism 310 platform (Applied Biosys-ems, Foster City, CA).

Figure 1. Pedigree shows COL4A3 mutations.

Figure 2. Schematic representation of the molecularembrane (GBM). (A) The GBM, located between podocy

ollagen that consists of protomers. (B) Each protomer, creriple helical collagenous domain, and a noncollagenousD) The NC1 domain is composed of 2 homologous subdo

496 and 1516 represent the nonsense mutations seen in our pati

Direct DNA sequencing in the older sister indicated 2eterozygous mutations in exon 49 of the COL4A3 gene:.4486C¡T (CGA¡TGA; p.Arg1496X) and c.4546C¡TCGA¡TGA; p.Arg1516X; Fig 2). Her younger sister hadhe same heterozygous mutations in COL4A3. In both sis-ers, heterozygous polymorphisms were detected in exons 7,, and 38, along with homozygous polymorphisms in exons1 and 25. Additional genetic testing of the parents showedhat their father was heterozygous for the p.Arg1496X

utation and their mother was heterozygous for the.Arg1516X mutation, confirming that these mutations weren trans. The family’s pedigree is shown in Fig 1. Subse-uent to the initial presentation of these results,3 DNAutations in these patients were reported online.4

DISCUSSION

Two novel mutations in the COL4A3 geneere found in African American siblings with

utosomal recessive Alport syndrome. They areompound heterozygotes, having inherited a del-terious mutation from each of their parents.oth siblings have symptomatic renal diseasend significant hearing deficits. However, theirarents, both carriers, are asymptomatic. Earlyevelopment of chronic kidney failure in combi-ation with deafness increasingly has been seenn the autosomal recessive form of Alport syn-

ization of type IV collagen in the glomerular basementendothelial cells, is formed by a suprastructure of type IV3 �(IV) chains, has a 7S domain at the N-terminal, a long

trimer at the C terminal. (C) Type IV collagen �3 chain.the first is shown here. The highlighted defects at position

organtes andated by(NC1)

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Novel Mutations in Alport Syndrome e27

rome, which accounts for only 15% of allorms. In many cases, autosomal recessive Al-ort syndrome is the result of consanguinity,4

lthough this was not the case in our family. Fewase reports described African Americans withlport syndrome; none were noted to be com-ound heterozygotes.5

There are approximately 30 different muta-ions resulting in autosomal recessive Alportyndrome, with the majority caused by COL4A4utations.6 Most cases report a missense muta-

ion causing a glycine substitution, which inter-eres with normal folding of the triple helicalrotomers. These missense mutations are locatedrimarily in the collagenous portion of the �3 or4 collagen chains. Our nonsense mutations,.Arg1496X and p.Arg1516X, were located inhe noncollagenous (NC1) domain of the COL4A3ene. An intact NC1 domain is necessary forormation of the collagenous triple helix neededor the �3�4�5 (IV) network that is essential inasement membrane structures. In our patients,eplacement of the arginine residue (Arg) createdremature stop codons (X), which presumptivelyead to a truncated protein. Subsequently, theollagen chain was shortened by approximately70 amino acids.Nonsense mutations are less common. How-

ver, Lemmink et al7,8 reported a case of aorwegian brother and sister who were com-ound heterozygotes for nonsense mutations inhe NC1 domain at the terminal end of the �3ollagen chain. There were other reported NC1omain defects typically resulting from eithereletions or splice-site mutations.8 Mutations inhe NC1 domain seem to differ clinically fromollagenous helix mutations with relation to de-ree of renal disease and severity and timing ofearing loss. Mutations at the terminal end of theene creating an early truncation appear to causeidney failure and sensorineural deafness at anarly age, as seen in our patients. However, theissense mutations of glycines in the collagen

elix cause deterioration in kidney function withnly mild or late-onset deafness after the thirdecade.9,10 In our case, because the youngeribling had the same genotype as the indexibling, it is predicted that she also will develophe same early clinical features of Alport syn-

rome. i

There are 12 conserved cysteine residues lo-ated in the NC1 domain that have been consid-red crucial in linking type IV collagen mol-cules together to form basement membranes.hether the conserved cysteine residues have a

ole in genotype-phenotype correlation is un-nown. If there is a role, based on our 2 patients,e propose that an individual must have more

han 4 intact cysteine residues to have a function-ng collagen basement membrane network. How-ver, additional subjects with premature chainerminations secondary to distal NC1 domainutations are needed to further substantiate this

heory.A few carriers with heterozygous mutations in

OL4A3 were observed to progress to chronicidney failure.11,12 However, there is a widepectrum of phenotypes in which some carriersre completely asymptomatic, whereas othersave persistent or intermittent microscopic hema-uria.13 In our case, the father, who was found toarry the p.Arg1496X mutation, is an asymptom-tic donor. He continues to have no evidence ofematuria or proteinuria since donor nephrec-omy. Since transplantation, the older sister hashown stable kidney function without protein-ria or hematuria. There is limited informationegarding the outcome of asymptomatic donors;ence, it is important to identify their carriertatus with close follow-up after transplanta-ion.14

In conclusion, we discovered 2 African Ameri-an siblings with novel mutations of exon 49 onhe COL4A3 gene resulting in Alport syndrome.hey are both nonsense mutations in the NC1omain of the �3(IV) collagen chain resulting inremature chain termination. The mechanism ofutations in the NC1 domain was reported in

nly 2 other families, neither African Ameri-an.7,8 Of these 2 patients, only 1 was a com-ound heterozygote. Only in our patient washere a documented family history negative foronsanguinity, which commonly accounts forutosomal recessive Alport syndrome. In an Afri-an American population in which autosomalecessive Alport syndrome is rarely seen, com-lete sequencing of the COL4A3 and COL4A4enes may be necessary to identify the underly-

ng mutation and confirm the diagnosis.

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ACKNOWLEDGEMENTSSupport: None.Financial Disclosure: None.

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OL4A4, and COL4A3 mutations in Alport syndrome. Humutat, Mutation in Brief #820 (2005) online. Available at:

ttp://www3.interscience.wiley.com/homepages/38515/pdf/820.df. Accessed March 20, 20085. Schatz H: Alport’s syndrome in a Negro kindred. Am J

phthalmol 71:1236-1240, 19716. Rana K, Tonna S, Wang YY, et al: Nine novel COL4A3

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OL4A3/COL4A4 mutations producing focal segmental glo-erulosclerosis in thin basement membrane nephropathy.Am Soc Nephrol 18:3004-3016, 200713. Gubler MC: Inherited diseases of the glomerular

asement membrane. Nat Clin Pract Nephrol 4:24-37,00814. Kashtan CE: The wages of thin. J Am Soc Nephrol

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