Stratified Whole Genome Linkage Analysis of Chiari Type I Malformation Implicates Known Klippel-Feil Syndrome Genes as Putative Disease Candidates

<div><p>Chiari Type I Malformation (CMI) is characterized by displacement of the cerebellar tonsils below the base of the skull, resulting in significant neurologic morbidity. Although multiple lines of evidence support a genetic contribution to disease, no genes have been identified. We therefore conducted the largest whole genome linkage screen to date using 367 individuals from 66 families with at least two individuals presenting with nonsyndromic CMI with or without syringomyelia. Initial findings across all 66 families showed minimal evidence for linkage due to suspected genetic heterogeneity. In order to improve power to localize susceptibility genes, stratified linkage analyses were performed using clinical criteria to differentiate families based on etiologic factors. Families were stratified on the presence or absence of clinical features associated with connective tissue disorders (CTDs) since CMI and CTDs frequently co-occur and it has been proposed that CMI patients with CTDs represent a distinct class of patients with a different underlying disease mechanism. Stratified linkage analyses resulted in a marked increase in evidence of linkage to multiple genomic regions consistent with reduced genetic heterogeneity. Of particular interest were two regions (Chr8, Max LOD = 3.04; Chr12, Max LOD = 2.09) identified within the subset of “CTD-negative” families, both of which harbor growth differentiation factors (GDF6, GDF3) implicated in the development of Klippel-Feil syndrome (KFS). Interestingly, roughly 3–5% of CMI patients are diagnosed with KFS. In order to investigate the possibility that CMI and KFS are allelic, GDF3 and GDF6 were sequenced leading to the identification of a previously known KFS missense mutation and potential regulatory variants in GDF6. This study has demonstrated the value of reducing genetic heterogeneity by clinical stratification implicating several convincing biological candidates and further supporting the hypothesis that multiple, distinct mechanisms are responsible for CMI.</p></div

Population characteristics.

a<p>Only considered genotyped individuals after exclusions were applied (See Methods section for details).</p>b<p>Mean +/− standard deviation [range].</p>c<p>Only considered affected individuals.</p><p>Abbreviations: CMI: Chiari Malformation Type I; No.: number.</p

Two-point and multipoint LOD scores obtained from stratified analysis.

<p>Only chromosomes with a maximum multipoint LOD score >2 are shown. LOD score thresholds of 2 and 3 are indicated by the blue and red lines, respectively. Green points and lines represent LOD scores under a linear model, blue points and lines represent HLOD scores, and red points and black lines represent LOD scores under an exponential model. CTD-positive families: Chr1 (A), CTD-positive families: Chr9 (B), CTD-negative families: Chr8 (C), CTD-negative families: Chr9 (D), CTD-negative families: Chr12 (E), and CTD-negative families: Chr17 (F). Negative two-point and multipoint LOD scores are set to zero. Plots were created in R 2.15.0.</p

Segregation of the missense mutation, rs121909352 (A249E), in two CMI pedigrees.

<p>Family 9453 (A) and Family 9476 (B). Symbols shaded in black indicate a diagnosis of CMI with or without syringomyelia, small diamonds represent a miscarriage, and symbols shaded in grey indicate an uncertain diagnosis. 9453-0001 has been diagnosed with a suspected Chiari Malformation Type 0 and 9476–1001 has been diagnosed with tonsillar ectopia. “+/+” indicates homozygous for the reference allele; “+/−“ indicates heterozygous for the variant allele. Sequences were generated in both the forward and reverse direction and are shown below each sampled individual. Progeny 8 (Delray Beach, FL) was used to construct the pedigrees and Sequencher 5.0 (Ann Arbor, MI) was used to create the chromatograms.</p

GDF6 and GDF3 selected sequence variants.^a

a<p>Only variants which were followed-up are shown here (See Methods section); Variants were validated by bidirectional sequencing and all sampled affected and unaffected individuals within each identified family were sequenced.</p>b<p>Base pair positions based on human genome build GRCh37/hg19.</p>c<p>The nomenclature used to describe novel variants was based on recommendations by the Human Genome Variation Society (den Dunnen and Antonarakis 2001). Nucleotide numbering was based on the GDF6 RefSeq genomic sequence, NG_008981.1, and intron-exon boundaries were defined based on the GDF6 mRNA sequence, NM_001001557.</p>d<p>Alleles: Reference allele/Alternate allele.</p>e<p>CMI MAF estimate based on all affected family members initially screened; 1KG MAF: Based on 1000 Genomes Integrated Phase 1 Release v3: European population.</p>f<p>Is sharing observed across all affected individuals within each family?</p>g<p>Numbers in parentheses: Numerator: number of sampled individuals carrying the variant, Denominator: total number of sampled individuals. Only affecteds were considered for “All affecteds” and only unaffecteds/uncertains were considered for “Reduced penetrance”.</p>h<p>MAF estimate was not available from 1000 Genomes; MAF estimate based on the Exome sequencing project: European population.</p>i<p>Individual suspected to have Chiari Malformation Type 0 is counted as “affected” for the purposes of this table.</p

Most significant two-point and multipoint LOD scores.^a

a<p>The top two most significant two-point results within each model and family subset as well as any maximum multipoint LOD score exceeding 2 are included.</p>b<p>When two markers are listed, the first corresponds to the marker used for the two-point result shown. The second corresponds to the closest marker included in the multipoint analysis.</p>c<p>LOD scores exceeding 2 are bold and LOD scores exceeding 3 are bold and italicized. For the parametric model, HLOD scores are shown.</p>d<p>Empirical p-values less than 0.05 are bold.</p><p>Abbreviations: CTD: connective tissue disorder, NPL: nonparametric linkage, LOD: logarithm of the odds, Emp: empirical, CW: chromosome-wide, GW: genome-wide, N/A: not applicable.</p