Novel nonsense mutation causes analbuminemia in a Moroccan family.
We describe a novel molecular defect causing analbuminemia in a 5-year-old girl, the first child of a couple from El Jadida, Morocco. The parents were cousins, and the mother, a healthy 31-year-old primigravida, had an uncomplicated pregnancy. At birth the child was observed to be "small for gestational age" (1735 g), but otherwise unremarkable. The placenta was edematous, weighing 960 g (54% of birth weight; normal <25%). Slight peripheral edema at 2.5 weeks triggered further investigations, leading to the diagnosis of analbuminemia. Plasma albumin was "low" by protein electrophoresis, <10 g/L by a routine chemical technique, and <6 mg/L by an immunoassay using polyclonal antibodies and kinetic nephelometry. Her plasma oncotic pressure in a recumbent position was low (12 mmHg; normal, 26-31 mmHg). After albumin infusion, the edema disappeared and did not recur, despite albumin concentrations again dropping below the detection limits of the assays. Both her physical and mental development were unremarkable (10). The parents' plasma contained decreased albumin (33 and 34 g/L, respectively) and marginally increased fractions of other proteins. Both had a low plasma oncotic pressure of 21 mmHg after 30 min in a recumbent position.
Genomic DNA from the proband and her parents was extracted from whole blood, obtained after informed consent, and the 14 coding exons of the human serum albumin gene and their intron-exon junctions (4) were PCR-amplified (8) with specific primer pairs (Table 1). Genomic DNA from two unrelated healthy volunteers was available as controls. All reactions were performed on a Hybaid thermocycler in a 25-[micro]L volume, using Ready to Go Beads (Amersham Pharmacia Biotech) with a final Mg[Cl.sub.2] concentration of 1.5 mM. Conditions for amplification with primers A13A and A14A included initial DNA denaturation at 94[degrees]C for 3 min, followed by 35 cycles of denaturation at 94[degrees]C for 30 s, primer annealing at 62[degrees]C for 30 s, and elongation at 72[degrees]C for 30 s. Final extension was performed at 72[degrees]C for 3 min. Primers A19B and A20C were used with the same protocol except for an annealing temperature of 64[degrees]C. For the other primers, the annealing temperatures ranged from 58 to 64[degrees]C. The PCR products, which ranged from 288 to 464 by in length, showed sharp bands when checked for homogeneity on a 3% agarose gel. The amplicons were mixed with equal amounts of single-strand conformation polymorphism (SSCP) buffer containing 950 mL/L formamide, 10 mmol/L NaOH, 2.5 g/L bromphenol blue, and 2.5 g/L xylene cyanol and then submitted to mutation screening by SSCP and heteroduplex analysis. An aliquot of each sample was denatured at 95[degrees]C for 3 min and cooled on ice before the electrophoretic separation. Denatured and nondenatured samples were then loaded on nondenaturing horizontal ultrathin (0.3 mm) 15% acrylamide (acrylamide/piperazine diacrylamide, 85:1) gels with a running buffer of 75 mL/L glycerol in 375 mmol/L Tris-formate buffer (pH 9.0); electrophoresis was performed in a Pharmacia Multiphor 11 apparatus at 8[degrees]C for 90 min at 0.8 W/cm. The electrodes consisted of paper wicks soaked in 1.04 mol/L Tris-borate buffer (pH 9.0), and the bands were visualized by silver staining (11).
[FIGURE 1 OMITTED]
The electrophoretic patterns indicated that the only clear change in both the homozygous and heterozygous samples, compared with controls, occurred in the 406-bp region encompassing exon 10 and the intron 9-exon 10 and exon 10-intron 10 junctions (Fig. 1A). The DNA from the parents (lanes 2 and 3) showed the presence of three bands corresponding to homo- and heteroduplex PCR products. The proband's sample (lane 1) showed only one band with a mobility similar to that of the controls (lanes 4 and 5). No variation attributable to conformation polymorphisms could be seen. The genomic DNA fragments from the patient, her parents, and the controls were gel-purified (QIAquick Gel Extraction Kit; Qiagen) and sequenced with the fluorescent dideoxytermination method (BigDye Terminator Cycle Sequencing Kit; Applied Biosystems Inc.) on an ABI 310 sequencer (Applied Biosystems Inc.). The results showed that the patient (Fig. 1B, electropherogram a) was homozygous for the insertion of a T in a stretch of eight Ts spanning positions 12086-12093 of intron 10 (4). The electropherograms from the father (Fig. 1B, electropherogram b) and the mother (data not shown) displayed a double sequence starting from nucleotide 12094, which indicated the presence of both the wild-type and the mutated alleles. These results are consistent with the inheritance of the trait. The mutation occurs 23 positions downstream the 5' splice site in a tract that does not contain conserved sequence elements. It is well known that splicing of the pre-mRNA transcript is a critical step and that this process requires control elements that may involve intronic sequences at ~150 by from either ends (12). However, on the basis of available literature data, no direct deleterious effect could be ascribed to the presence of an additional T in this polyT tract.
We next examined the possibility of other changes that might have escaped electrophoretic differentiation. Data collected from different laboratories indicate that the detection rate by SSCP analysis decreases for DNA fragments longer than 200 by (13). Therefore, the PCR products were digested with the appropriate restriction enzymes (Table 1), and the fragments were analyzed using the same electrophoretic protocol. After digestion with StyI, a difference became evident within the region amplified with PCR primers A13A and A14A. This 394-bp long fragment was cleaved into two fragments of 266 and 128 bp, respectively. The results of the SSCP analysis of the digest are shown in Fig. 1C. The proband's sample (lane 1) showed a slight difference that was not detectable in DNA samples from the controls (lanes 4 and 5) or her parents (lanes 2 and 3). This result suggested that the region encompassing exon 7 and the intron 6-exon 7 and exon 7-intron 7 junctions from the proband contained a mutation; it therefore was sequenced together with the amplicons from her parents. The electropherogram of the proband showed that she is homozygous for a G [right arrow] T transversion at nucleotide 7796 in exon 7 (4) (Fig. 1D, electropherogram a). The mutation changes codon GAA for Glu244 to the stop codon TAA, leading to a premature termination of the polypeptide chain. The putative protein product would have a length of only 243 amino acid residues instead of the normal 585 found in the mature serum albumin. The DNA samples from her father (Fig. 1D, electropherogram b) and mother (data not shown) revealed a double peak at position 7796 attributable to the presence of both the wild-type and the mutated alleles. This result indicates that the trait is inherited.
DNA sequencing is widely accepted as the most sensitive method for the identification of genetic alterations, but it is impractical for analyzing large numbers of samples on a routine basis. Thus, numerous mutation detection methods have been developed, and among these, the most widely used is the electrophoretic analysis of heteroduplexes and SSCP. This combination allows the localization of genetic alteration within a given region with high sensitivity (13). In this study, we analyzed all 14 exons of the human serum albumin gene and the flanking intron regions, and heteroduplex analysis revealed the mismatch produced by a single-base insertion. This result is in agreement with the view that the differential separation of heteroduplexes and homoduplex DNA is greater when the sequence difference is an insertion or a deletion compared with a single-base substitution. Digestion of the PCR products with restriction enzymes improved the sensitivity of SSCP analysis, and the G-to-T substitution responsible for the analbuminemic trait was identified. Many factors are known to affect the reproducibility of SSCP analysis by slab gels, including temperature and DNA quantity and purity, and might explain the observation that no clear band shift was detected in the DNA samples from the heterozygous parents.
We thank Dr. Hugo Monaco for critical reading of the manuscript. This work was supported by Progetto di ricerca di interesse nazionale (PRIM) grant "Structural studies on hydrophobic molecule-binding proteins" from the MIUR, Ministero dell' Istruzione, della Universita e della Ricerca (Rome, Italy) and by the Cariplo Foundation.
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Monica Campagnoli,  Alberto Sala,  Assunta Romano,  Antonio Rossi,  Jeroen Nauta,  Bart G.P. Koot,  Lorenzo Minchiotti,  and Monica Galliano  *
 Department of Biochemistry "A. Castellani", University of Pavia, Pavia, Italy;  Department of Pediatric Nephrology, Erasmus MC, Sophia Children's Hospital, Rotterdam, The Netherlands; * address correspondence to this author at: Department of Biochemistry "A. Castellani", University of Pavia, viale Taramelli 3B, 27100 Pavia, Italy; fax 390382-423108, e-mail firstname.lastname@example.org
Table 1. Restriction enzymes used to digest the PCR-amplified exons of the human serum albumin gene. Fragment size, Primers (a) (b) bp Restriction Exon Forward Reverse enzyme Before After 1 A01A A02A HincII 464 252/212 2 A03A A04A DraI 288 159/129 3 A05A A06A AluI 356 182/174 RSaI 211/145 4 A07A A08A DdeI 381 191/190 5 A09A A10A MspI 309 182/127 6 A11A A12B DraI 396 216/180 RSaI 237/159 7 A13A A14A StyI 394 266/128 8 A15B A16B Sau3AI 399 297/102 9 A17A A18A XbaI 416 229/187 10 A19B A20C DraI 406 280/126 RSaI 210/196 11 A21A A22A HincII 343 209/134 12 A23A A24A StyI 386 264/122 13 A25C A26B StyI 381 207/174 14 A27A A28A SspI 339 204/135 (a) Primer sequences are given in Watkins et al. (8). (b) Sizes of PCR-amplified fragments before and after restriction enzyme digestion.
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|Title Annotation:||Technical Briefs|
|Author:||Campagnoli, Monica; Sala, Alberto; Romano, Assunta; Rossi, Antonio; Nauta, Jeroen; Koot, Bart G.P.;|
|Date:||Jan 1, 2005|
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