Molecular Analysis of the ABCA4 Gene Mutations in Patients with Stargardt Disease Using Human Hair Follicles

ABCA4 gene mutations are the cause of a spectrum of ABCA4 retinopathies, and the most common juvenile macular degeneration is called Stargardt disease. ABCA4 has previously been observed almost exclusively in the retina. Therefore, studying the functional consequences of ABCA4 variants has required advanced molecular analysis techniques. The aim of the present study was to evaluate whether human hair follicles may be used for molecular analysis of the ABCA4 gene splice-site variants in patients with ABCA4 retinopathies. We assessed ABCA4 expression in hair follicles and skin at mRNA and protein levels by means of real-time PCR and Western blot analyses, respectively. We performed cDNA sequencing to reveal the presence of full-length ABCA4 transcripts and analyzed ABCA4 transcripts from three patients with Stargardt disease carrying different splice-site ABCA4 variants: c.5312+1G>A, c.5312+2T>G and c.5836-3C>A. cDNA analysis revealed that c.5312+1G>A, c.5312+2T>G variants led to the skipping of exon 37, and the c.5836-3C>A variant resulted in the insertion of 30 nucleotides into the transcript. Our results strongly argue for the use of hair follicles as a model for the molecular analysis of the pathogenicity of ABCA4 variants in patients with ABCA4 retinopathies

Characteristic features on ophthalmological evaluation in the proband from family A.

<p>Fundus examination showed bilateral temporal pallor of the optic disc. PVEP presented a reduced amplitude and delayed latency of P100 wave. Components of PVEP, N75, P100 and N135, indicate polarity (negative or positive) and absolute latencies (msec) of the peaks waveforms. Right panel—left eye, left panel—right eye.</p

Quantification of the <i>OPA1</i> mutant transcripts.

<p>Quantification of the <i>OPA1</i> mutant transcripts.</p

Identification of a novel p.Q31* <i>OPA1</i> mutation.

<p>Pedigrees of the investigated ADOA family A (A) and B (C). Probands are indicated by arrows. Black symbols denote affected subjects, white symbols denote unaffected subjects without <i>OPA1</i> mutation. Symbols with a black dot inside refer to healthy mutation carriers. Bolded numbers next to the symbols denote the respective DNA samples (AC). Marker names (analyzed for both families) are shown on the left of the family A pedigree. Unbolded numbers to the right of the marker names denote microsatellite repeat motif sizes. The grey rectangle marks the location of the <i>OPA1</i> gene within the 3q28–29 region (AC). Wt/wt refers to patients homozygous for the wild-type allele, while */wt to patients heterozygous for the p.Q31* <i>OPA1</i> mutation (AC). Electropherogram from Sanger bidirectional sequencing of the <i>OPA1</i> exon 2 shows the c.91C>T (CAA>UAA) heterozygous mutation, predicting a premature stop codon p.Q31* in the OPA1 protein (B).</p

<i>OPA1</i> expression at mRNA level.

<p><i>OPA1</i> mRNA level were quantified in PBMC in patients with p.Q31* mutation and control samples from healthy individuals. The amount of <i>OPA1</i> mRNA was quantified by real-time PCR in relation to <i>GAPDH</i>. The values of control samples were set at 1.</p