Page 59 - Haematologica May 2022

FGG mutation in congenital afibrinogenemia
ence of an insertion of 36 bp in exon 8 of the FGG gene NM_021870.3,exon8,c.959_960insAATCCACCTGCTG CAAAATATCCAGTAGTTTGGCAT. This abnormality was present in only 14.2% of the reads (14/98) covering this position. The apparent localization of the insertion in FGG exon 8 was in contradiction with our previous results demonstrating the absence of a causative mutation in any fibrinogen gene coding sequence. BLAT analysis revealed that the 36 bp-long sequence was in fact in intron 8 of FGG suggesting the presence of a more com- plex local rearrangement. The visual inspection of the reads aligned to FGG exon 8 further supported this hypothesis with the presence of hard clipped and out- ward-facing read pairs spanning far apart from that expected based on the library insert size (Figure 2). This configuration of the read pairs was highly suggestive of the presence of a heterozygous duplication that could not be fully characterized by our exon-centered analysis.
We therefore amplified by PCR a larger portion of FGG from intron 7 to exon 10 which yielded a 4 kb prod- uct corresponding to the normal sequence and a 4.4 kb product for affected individuals. Heterozygous individu- als showed both bands, as expected (Figure 3A). Sequencing of the larger band revealed the presence of a duplicated sequence of 403 bp (out of 404, one base in a stretch of 4 intronic “A”s is missing) containing the last 169 bases from FGG exon 8, the donor splice site GT, and 232 additional bases of FGG intron 8 (Figure 3B). Genotypes for all family members are indicated in Table I. The presence of the duplication in homozygosity was associated with absence of fibrinogen in circulation and afibrinogenemia in all four affected individuals, while heterozygosity was associated with decreased fibrino- gen levels (Table I).
Duplication causes aberrant splicing of both FGG transcripts
Because the mutation duplicates the donor splice site of intron 8, we predicted that the impact of the mutation would be on FGG transcript splicing. Interestingly, two FGG transcripts are normally produced which differ at the 3’end: the major g chain mRNA has ten exons while in the minor g’ chain isoform intron 9 is not spliced out, substituting the four amino acids encoded by exon 10 with twenty g’ COOH-terminal residues.19-21 The pres- ence of the duplication was thus anticipated to affect splicing of both isoforms. HEK-293T cells were transient- ly transfected with minigene constructs encompassing intron 7 to exon 10 with and without the duplication (Figure 4A). RNA produced were reverse transcribed to cDNA which were used in two different PCR reactions to amplify transcripts containing exon 10, present in the major g transcript, and transcripts containing the last bases of exon 9, retained in the minor g’ transcript but spliced out in the major g transcript. The results obtained for transcripts containing exon 10 (Figure 4B) showed one major product, indicated by an asterisk, for the normal minigene which was shown by sequencing to correspond to the correctly spliced mRNA containing exons 8, 9 and 10 encoding the major g chain. Sequencing of clones of individual PCR products showed that a transcript retain- ing intron 9 was also produced, thus encoding the minor g‘ transcript even though exon 10 is present. For the mutant, the major product obtained, indicated by an asterisk, retained intron 8 with the duplication, resulting
in a transcript with a frameshift and a premature truncat- ing codon 13 codons downstream.
Cloning of the PCR products allowed the identification of additional minor aberrant transcripts resulting in frameshifts and premature truncating codons. One includ-
Table 1. Patient symptoms, fibrinogen measurements and genotypes.
Patient ID
Fibrinogen Fibrinogen Bleeding clauss antigen score
(mg/dL) (mg/dL)
197 193 0 273 204 0 282 254 0 169 215 0 214 218 0 231 206 0 <20 <2 3 213 209 0 105 150 0 152 177 0 85 126 0 152 170 1 226 260 0 291 322 0 170 161 0 103 122 0 115 110 2 130 145 1 273 320 0 144 142 0 186 175 0 317 334 0 246 267 0 n.d. n.d. 0 272 260 0 334 342 0 90 86 2 152 157 0 269 267 0 108 132 1 441 448 0 266 248 0 203 192 0 389 450 0 222 216 0 123 161 0 149 187 0 <20 <2 3
157 0 <2 3 <2 3 350 0 140 1 220 1 171 0
Thrombosis
Yes
Genotype
normal normal normal heterozygous normal normal homozygous normal heterozygous heterozygous heterozygous heterozygous normal normal normal heterozygous heterozygous heterozygous heterozygous heterozygous normal normal normal normal normal normal heterozygous heterozygous normal heterozygous normal normal normal normal normal heterozygous heterozygous homzygous heterozygous homzygous
1277
1278
1279
1280
1281
1282
1283
1284
1285
1286
1287
1288
1289
1290
1291
1292
1293
1294
1295
1296
1297
1298
1299
1300
1301
1302
1303
1304
1305
1306
1307
1308
1309
1310
1311
1312
1313
1314
1315 127 1316 <20 1317 <20 1318 338 1319 121 1320 179 1321 165
Yes homzygous normal
heterozygous normal heterozygous
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