Clinical and Molecular Study of the Extracellular Matrix Protein 1 Gene in a Spanish Family with Lipoid Proteinosis
The index patient was a 35-year-old woman born to unrelated parents who developed a progressively hoarse voice from 9 months of age, and vesicular and bullous lesions that appeared after minimal trauma. A physical examination revealed deposition of hyaline material in the larynx, epidermolysis bullosa, multiple yellowish warty skin papules on the body, and diffuse acneiform scars over her face, with blisters in the mouth, eyes, and areas subject to friction. Neurological symptoms included unilateral paresthesia, dizziness, and memory loss associated with depression, in addition to a decline in verbal fluency and impaired coordination of movement from the age of 19 years. Computed tomography revealed bilateral and symmetric amygdaloid-uncal calcifications (). Gradient refocused-echo T2*-weighted MRI images () revealed marked hypointense bilateral rounded symmetric lesions in the mesial temporal lobes, involving the amygdaloid complex. A routine EEG (awake and sleep) recording was slightly unstable, but no epileptiform activity was evident. A cognitive event-related potential P-300 wave revealed a reaction-time delay during development of the Posner task, with a normal latency of the P3 component during the oddball task. The results of a neurological examination were unremarkable, and a neuropsychological evaluation revealed a decrease in information processing speed and disturbances in episodic memory and the amnesic evocation process. These findings could be explained by her depressed mood. The results of a blood workup were unremarkable.\nA skin biopsy () of the granuloma annulare revealed sweat glands and small nerves with deposition of strongly periodic-acid-Schiff-positive homogeneous, eosinophilic, hyaline-like material and diastase resistant granules around dermal blood vessels, revealing its glycoproteic nature, which is consistent with LP.\nGenomic DNA was purified from blood samples using a salting-out procedure, and stored in TE buffer. ECM1 was amplified by a polymerase chain reaction (PCR) with primers spanning all ten exons and intronic flanking regions. Genomic DNA (100 ng) was amplified in 20-µL reaction volumes containing 20 pmol of primers (), 1.5 nmol of MgCl2, 4 nmol of dNTP, and 1 U of AmpliTaq DNA polymerase (Applied Biosystems, Warrington, UK). The PCR products were separated by 1.5%-agarose-gel electrophoresis and purified with the Qiaquick Gel Extraction Kit (Qiagen, Crawley, UK). Amplified fragments were run and sequenced in an ABI 3130 genetic analyzer.\nSequencing of exon 7 revealed the presence of a single base substitution at position 1076 of cDNA (GenBank accession no. NM_004425.3). The c.1076G>A transversion causes a premature termination codon, p.Trp359*, that affects the Ecm1a isoform. The patient was found to be homozygous for the mutation, while her parents were heterozygous carriers (). No other changes or polymorphisms were found in the ECM1 coding region. The mutation causes the loss of a restriction target for NlaIII endonuclease, which was therefore used as a screening test. The mutation was not present in 106 chromosomes of unrelated control subjects.\nFour different polymorphic dinucleotide microsatellites were analyzed in this study: D1S2344, D1S2345, D1S305, and D1S2624. The primer sequences were obtained from the uniSTS database of the National Center for Biotechnology Information. The sizes of the microsatellites were determined by direct sequencing in the ABI 3130 genetic analyzer.\nThe four markers allowed determination of the segregation phase and the haplotypes associated with the c.1076G>A transversion. The parents' haplotypes differed significantly in the mutated chromosomes, although double recombination events cannot be ruled out ().