Biologic relationships and gender assignment of the family members were confirmed using the ABI profiling kit. Despite age induced degradation of the DNA samples extracted from Patient 2’s histology slides, 8 of the 10 loci included in the kit amplified. As expected with sample degradation, the largest of the STR markers (D18S51 and D7S820) failed to amplify, but the gender was confirmed by the amelogenin marker (data not shown).
Using primers and conditions included in supplemental Table I
, the same de novo germline HRAS
mutation c.34G>A, predicting a p.G12S amino acid substitution, was discovered in every DNA sample isolated from Patient 1 and his affected sister, Patient 2 (). This mutation was absent in all parental DNA samples.
Figure 4 Detection of the germline HRAS mutation in two siblings. The sequence alignments around nucleotide c.34 are shown in the patients and parents. Patient 1’s buccal DNA and Patient 2’s archived liver or kidney DNA samples display the c.34G>A (more ...)
In an attempt to identify the parental origin of the mutation in both affected siblings, the family members were genotyped across a ~3.8 kb region of the HRAS locus (). Six known polymorphic sites, including three single nucleotide polymorphisms (SNPs, rs8176330, rs12628 and rs35601764) and three short tandem repeats (STRs, rs112488103, rs113931482 and rs112587690) were used in this study. During our ongoing molecular study of Costello syndrome and survey of the HRAS locus, linkage disequilibrium (LD) was found in 103/106 individuals for rs8176330, rs112488103, and rs35601764. A second LD group was identified in 287/287 genotyped individuals that included markers rs112587690 and rs12628 (data not shown).
Figure 5 HRAS locus: Schematic representation of the genomic structure of the HRAS gene including the mutation site c.34 G>A and location of six polymorphic sites, as indicated above the sequence by their rs number and alleles. Untranslated exon 1 and (more ...)
Genotype data for the family at the HRAS locus () showed that Patient 1 and his father were homozygous at five sites and heterozygous 8/6 at rs113931482. Interestingly, the mother was heterozygous at all six sites. Due to the level of degradation of Patient 2’s DNA samples, direct genotyping information was obtained for only three of these polymorphic markers. However, based on the LD patterns, Patient 2’s genotype could be inputted at the three missing sites rs8176330, rs112587690 and rs35601764 (), making both siblings identical at all six polymorphic sites. Additional sharing of maternal alleles was observed at 11p using linkage mapping set markers: Of six 11p loci genotyped, both siblings shared four while the other two loci were uninformative (data not shown).
Family Genotyped at Six Polymorphic Sites
As seen in , the only heterozygous site available to determine mutation parental origin in Patient 1 was the 8/6 repeat region of rs113931482. shows the double sequence profiles typically seen in 8/6 heterozygous individuals carrying one allele with six copies and one allele with 8 copies of the basic CGC repeat, in this case Patient 1 and his mother. In order to determine which of these alleles also carried the mutated c.34A base, allelic specific amplification was used on Patient 1 DNA. During allelic specific amplification (ASA), each individual chromosome is amplified separately and genotypes can resolve into two distinct haplotypes. displays the result of ASA performed with either the mutated (Mut c.34A) or wild type (WT c.34G) allele and clearly indicates that the mutation in Patient 1 originated on the chromosome carrying eight copies of the GCG repeat, while the wild type allele carries six copies. Whether the 8- or 6-copy allele was contributed by the maternal germ cell was determined by ASA using SNP rs12628 allele specific primers (c.81T or c.81C) to generate haplotype information for the Patient’s mother. The sequencing results presented in show that the 81C allele was on the same chromosome as the six GCG repeats, and that the 81T allele which she passed on to Patient 1 (see ) carried eight copies of the GCG repeat. Since Patient 1’s mutation was on the allele carrying the eight repeats, we conclude that his Costello syndrome causing mutation is maternally inherited (). His father contributed the second 81T allele, the wild type HRAS c.34G allele, as well as six copies of the GCG repeat (). The degree of allele sharing between the siblings at the HRAS locus and along 11p suggested that Patient 2’s mutation was also maternally inherited. However, her degraded DNA was not a suitable template for ASA amplification of large amplicons.
Figure 6 Sequencing of the region around rs113931482. This family is heterozygous 8/6 at this site () and the double sequencing profiles observed in Patient 1 and his mother are typical of heterozygous individuals carrying alleles with six or eight copies (more ...)