A Novel Locus for Adolescent Idiopathic Scoliosis on Chromosome 12p

Abstract

Adolescent idiopathic scoliosis (AIS) is a common disorder with strong evidence for genetic predisposition. Quantitative trait loci (QTLs) for AIS susceptibility have been identified on chromosomes. We performed a genome-wide genetic linkage scan in 7 multiplex families using 400 marker loci with a mean spacing of 8.6 centiMorgans (cM). We used Genehunter Plus to generate linkage statistics, expressed as homogeneity (HLOD) scores, under dominant and recessive genetic models. We found a significant linkage signal on chromosome 12p, whose support interval extends from near 12pter, spanning approximately 10 million bases or 31 cM. Fine mapping within the region using 20 additional markers reveals maximum HLOD = 3.7 at 5 cM under a dominant inheritance model, and a split peak maximum HLOD = 3.2 at 8 and 18 cM under a recessive inheritance model. The linkage support interval contains 95 known genes. We found evidence suggestive of linkage on chromosomes 1, 6, 7, 8, and 14. This study is the first to find evidence of an AIS susceptibility locus on chromosome 12. Detection of AIS susceptibility QTLs on multiple chromosomes in this and other studies demonstrate that the condition is genetically heterogeneous.

INTRODUCTION

The Scoliosis Research Society defines adolescent idiopathic scoliosis (AIS) as a lateral spinal curvature ≥10° on plane radiographs, developing after age 11 and without any identifiable underlying secondary cause. AIS is a common condition, estimated to affect 2%–3% of the population.¹ There is a 2:1 female preponderance of scoliotic curves exceeding 10°, but the incidence of smaller curves is equal in males and females.

Manifestations of AIS and its treatment are manifold and life-threatening when curves exceed 70°.^2,3 Management varies according to curve magnitude.⁴ Treatment for patients with small curves (<25°) is observation until maturity. Currently, the standard of care for curves between 25° and 45° in a growing patient requires use of an underarm thoraco-lumbar-sacral brace worn 22 hours per day.⁵ Curves exceeding 45° in a growing child require surgical intervention, consisting of posterior spinal fusion and/or anterior fusion and instrumentation.

Various hypotheses regarding AIS pathogenesis have been proposed,^6–10 with multifactorial causation the current majority opinion. In spite of limited insight into mechanism, there is strong evidence that genetic predisposition contributes to AIS. Several research groups have identified quantitative trait loci for scoliosis to various autosomes and to the X chromosome.^11–18

We undertook linkage mapping of seven previously unreported families with multiple AIS-affected individuals. We found evidence of a novel AIS susceptibility locus on chromosome 12p. The chromosome 12 susceptibility locus is detected under both dominant and recessive models, and is localized to a 9 million base region harboring approximately 100 known genes.

MATERIALS AND METHODS

Members of 7 multigeneration, multiplex AIS families of European ancestry were recruited at the Hospital for Special Surgery, New York, New York. Clinical phenotype was validated by clinical evaluation and radiographic analysis. AIS was diagnosed if onset of scoliosis was age 11 years or greater, there was no identifiable secondary cause of scoliosis, and there was documentation of ≥10° spinal curvature by the Cobb method. All subjects enrolled in the study had a complete physical examination by two investigators (CLR [pediatric orthopedist] and PFG [clinical geneticist]). Radiographs were performed when the angle of trunk rotation measured by scoliometer was 5° or greater, as is the current standard of clinical practice. None of the patients had skeletal evidence of Marfan syndrome. All subjects provided informed consent/assent, and the study was Institutional Review Board-approved.

DNA was prepared by standard methods from whole blood. The whole genome linkage scan was performed using the Marshfield screening set 16, which includes 400 microsatellite markers with average spacing of 8.6 cM (details available at http://research.marshfieldclinic.org/genetics/sets/combo.html). Genotyping was performed as described previously.¹⁹ Purpose-developed software packages called SCORER and IMAGER were used for allele calling, genotype checking, and data management.²⁰ Preliminary data integrity checks were performed to verify the Hardy-Weinberg equilibrium of each marker, identify and exclude contiguous regions of homozygosity, incompatible genotypes, gender errors, marker dropout, and individual dropout.

We performed our primary parametric linkage analysis with Genehunter Plus^21,22 under both dominant and recessive inheritance models. Other model assumptions included a population AIS frequency between 1% and 3% and penetrance between 0.60 and 1.00. We used PEDCHECK²³ and RELPAIR²⁴ to confirm consistency with Mendelian inheritance and to trace and correct errors. Single-point and multi-point analyses were carried out for the 7 families together and homogeneity LOD (HLOD) score was determined according to combined recombination information and heterogeneity within the families. A heterogeneity parameter was calculated in GENEHUNTER to indicate the probability of the same phenotype resulting from different genes or alleles. Loci with HLOD score higher than 2.3 and 3.4 were considered as suggestive and significant for linkage.²⁵ Fine structure mapping with 20 additional microsatellite markers was chosen on the basis of information and even spacing within the candidate region.

The first secondary analysis omitted families 1 and 5, which were the outliers in the heterogeneity analysis. The second included all 7 families and was performed using Superlink v. 1.6.^26,27

RESULTS

The study population (Fig. 1) included 54 individuals, 21 of whom had AIS by the above criteria and 2 more of whom had spinal curvature that approached but fell short of the 10° threshold for affected status and were there fore classified as unaffected in the linkage analysis.

Figure 1.

Pedigrees of families studied. Management was determined by curve magnitude. Curves ≤25° were observed (O), curves between 25° and 40° were treated with bracing (B), and curves >40° were surgically corrected (S). Circles = females. Squares = males. Shaded circles or squares indicate affected. Index cases are indicated by an arrow.

The results of the best model’s genome-wide linkage analysis are displayed in Fig. 2. A significant linkage signal, with HLOD = 3.5 with homogeneity (α = 1.0), disease frequency of 0.02, and penetrance of 0.99 was observed at GATA49D12 under a recessive model. We observed several other linkage signals with HLOD >1.0, including chromosome 1 (marker GTTTT002P: HLOD [recessive] = 1.7), chromosome 6 (marker ATA6C09P: HLOD [dominant] = 1.1), chromosome 7 (marker GATA24F3ZP: HLOD [recessive] = 1.2), chromosome 8 (markerUTZ129L: HLOD [recessive] = 1.2), chromosome 12 (marker D12S372: HLOD [recessive] = 2.0; HLOD [dominant] = 1.5), chromosome 14 (marker GGAA30H04ZP: HLOD [recessive] = 2.0). The HLOD maximum in the dominant genome-wide scan was 1.5 at GATA4H03.

Figure 2.

Genome-wide linkage profiles with dominant and recessive models. The length of whole genome consists of all 24 chromosomes sequentially and is represented in centiMorgans (cM). The profile in red stands for the result of recessive model, while the black one represents the dominant model. HLOD is the homozygous LOD score which combines multi-family information based on homogeneity of all families at every locus.

To further investigate the significant linkage signal at GATA49D12, we performed fine structure linkage mapping of chromosome 12p with an additional 20 microsatellite markers listed in Table 1. The results are shown in Fig. 3. The dominant model yields a linkage peak with HLOD = 3.7 (α = 1.0) at 5 cM, while the recessive model reveals a pair of peaks with HLOD = 3.2 (α = 1.0) at 8 and 18 cM. The “pair” of recessive linkage peaks more likely represents a single locus at 13 cM.

Table 1.

Markers Used for Fine Mapping

Marker	Position (bases)	HLOD recessive	HLOD dominant	Source
D12S341	719,580	0.2	0.8	deCode
D12S94	780,851	0.3	0.1	deCode
D12S1608	1,629,617	0.6	1.7	deCode
D12S1656	1,677,650	0.8	0.1	deCode
D12S100	2,046,914	2.6	3.7	deCode
D12S1615	2,640,746	1.7	0.7	deCode
D12S1626	3,165,986	2.6	1.2	deCode
D12S372	3,457,710	2.3	1.5	Mfld screen set 16
D12S1652	3,583,169	3.2	1.5	deCode
D12S1685	3,862,066	3.2	1.1	deCode
D12S1725	4,315,907	1.3	1.3	deCode
D12S1624	4,581,357	2.3	2.3	deCode
D12S314	4,838,740	1.7	0.6	deCode
D12S1623	6,792,183	2.3	0.3	deCode
D12S1625	6,975,843	2.9	0.3	deCode
GATA49D12N	8,048,975	3.2	0.0	Mfld screen set 16
D12S1695	9,046,784	2.4	0.0	deCode
D12S1674	10,087,431	1.9	1.1	deCode
D12S1697	11,685,667	1.9	0.0	deCode
D12S89	11,793,428	1.9	0.0	deCode

Figure 3.

Fine mapping results of 12p3 region. Both physical and genetic distances from 12pter are given. Single-point analysis yields both LOD and HLOD of 3.2 at 12p (GATA49D12N). The heterogeneity parameter alpha is 1.0. NPL score is 1.38 with p-value of 0.053. The dominant model yields a linkage peak with HLOD = 3.7 (α = 1.0) at 5 cM, while the recessive model reveals a pair of peaks with HLOD = 3.2 (α = 1.0) at 8 and 18 cM. Marker positions are those provided by deCode. The exact chromosome position can be found in Table 2. The genetic and physical distances are not linearly related due to varying recombination rates along the length of the chromosomes. Moreover, there are discrepancies in marker order between the genetic and physical maps shown as boldfaced physical map position labels.

We performed a heterogeneity analysis to estimate how many families contributed to the observed linkage signals on chromosome 12p. For the recessive model, we found that α = 1, indicating that all the families contribute to the linkage signal. For the dominant model, α ranges between 0.3 and 0.8, with families 2, 3, 4, 6, and 7 contributing to the linkage signal. Secondary analyses, restricting the linkage analysis omitting families 1 and 5, and using Superlink gave virtually identical results (data not shown).

The candidate interval extending from D12S1608 at 1,677,650 base pairs from pter to D12S1674 at 10,087,431 base pairs, corresponding to the 12p13.3 cytogenetic band. The region is gene rich and includes 95 known genes and a comparable number of uncharacterized known and potential transcripts.

DISCUSSION

We report significant evidence of linkage of AIS to chromosome 12p based on data from 7 newly reported multiplex kindreds. By limiting the study to families in which there is more than one affected member, and by choosing families with at least one member with a curve exceeding 25°, we have designed our study to maximize the chance of finding an oligogenic inheritance pattern.²⁸ Moreover, our sample displays a high degree of homogeneity, with all the families contributing to the observed recessive linkage and 5 of the 7 families contributing to the observed dominant linkage. While none of the pedigrees appears to fit an autosomal recessive mode of inheritance, a pseudodominant mode of inheritance is possible, which can result in an affected offspring through the union of a homozygous and a heterozygous individual. We hypothesize an additive model of inheritance with contributions from both dominant and recessive models. The data do not allow us to determine whether the same locus is being found under both recessive and dominant models, or to establish whether the linkage peak represents a single locus or several tightly linked loci. Although we do not believe that any of our families are consanguineous, all are of European descent and have resided in the New York metropolitan area, so it is possible that some share common ancestors. Investigation of 7 unrelated southern Chinese families in Hong Kong revealed a significant major AIS susceptibility gene at 19p13.3.¹² As in our study, although the families were apparently unrelated, common ancestry could not be excluded. Occult consanguinity was reported in a genealogical study of AIS families recruited in Salt Lake City, in which 127 of 131 apparently unrelated probands were found to share common ancestry.²⁹ Moreover, the Salt Lake City kindreds shared significantly more common ancestry than a control cohort matched by birth year.

Several other research groups have studied AIS genetics (Table 2). The most progress has been made on chromosome 8, where CHD7¹⁸ and SNTG1³⁰ have been proposed as candidate genes. Previous work has also suggested the possibility that genes contributing to AIS may, when mutated, be responsible for known genetic syndromes. CHD7 mutations are responsible for the CHARGE (coloboma of the eye, heart defects, atresia of the choanae, retardation of growth and/or development, genital and/or urinary abnormalities, and ear abnormalities) syndrome.³¹ The chromosome 17 locus includes a region duplicated in Charcot-Marie-Tooth type 1A syndrome.¹³ Scoliosis occurs in approximately one third of patients with Charcot-Marie-Tooth, likely secondary to a neuromuscular etiology.³²

Table 2.

Summary of Prior AIS Linkage Studies

Reference	N (families/subjects)*	Location	Methods	Comments
18	52/123 (affected)	1p, 8q, 10q	“Affecteds only” nonparametric linkage, TDT	CHD7 identified as candidate gene on chromosome 8
16	202/1198	1,6,8,9,16 and17	Genomic screen and model independent analysis	As threshold for scoliosis curvature increased, chromosomes 9 and 16 became more significant
17	202/1198	5p13, 13q13.3, 13q32	Nonparametric sib pair, linkage disequilibrium	Kyphoscoliotic subgroup analysis
11	1/14	6q distal 10q 18q	Two point parametric and nonparametric multipoint linkage	Genome wide search in one family (7 affected members), followed by testing of “hot spots” in a second large family
13	1/17	17p11	Parametric linkage	Overlaps duplication in CMT type 1A
12	7/52	19p13.3	Parametric and nonparametric linkage	Recruited patients in whom scoliosis developed in adolescence
15	202/1198	19p13	Nonparametric sib pair, parametric linkage	Subgroup analysis limited to kindreds in which at least 1 member had curve ≥30°
14	202/1198	Xq23, Xq26.1	Nonparametric sib pair, parametric linkage	Estimate that 15% of AIS families display X-linkage
41	25/116(affected)	9q31.2-q34.2; 17q25.3-qtel	Two point parametric and nonparametric multipoint	Confirmation of ref. 15

^*NR = not reported.

Taken as a whole, the existing literature points to genetic heterogeneity, with multiple genes involved in AIS pathogenesis, and to oligogenic causation, with a major gene segregating in most affected families. Both these generalizations are conditioned in part by methodology, reflecting recruitment of distinct populations and more severely affected subjects.

The candidate region for the chromosome 12 susceptibility locus is gene rich, including 95 known genes and a similar number of uncharacterized known or potential transcripts. Some of the genes in the interval are attractive candidates for AIS susceptibility. These include genes encoding ion channels (KCNA1,³³ KCNA5,³³ KCNA6,³³ SCNN1A³⁴), bone derived growth factors (GDF3),³⁵ growth factors regulating muscle (FGF6)³⁶ and neurons (NTF3),³⁷ enzymes involved in protein processing (USP5,³⁸ SPSB2³⁹), and components of the extracellular matrix (MFAP5).⁴⁰ Their participation in neuromuscular and connective tissue development makes these plausible positional candidate genes.

The strength of linkage mapping as an investigative strategy is that it allows us to identify candidate genes by genomic location, without prior knowledge of function. However, our study also has important limitations. Our findings are based on a small number of families. Given ample evidence of the genetic heterogeneity of AIS, it is uncertain how frequently our findings can be generalized to other affected kindreds. The large number of genes included within the candidate interval is another important limitation of our study. Further fine mapping studies in additional families will ultimately allow the interval to be further refined and limited, reducing the number of potential candidate genes.

In spite of recent progress in AIS genetics, we know little regarding the developmental processes that operate during adolescent axial skeletal growth. Limited mechanistic understanding prevents us from predicting which patients will have progressive disease as they mature. Discovering the mechanisms that mediate vertebral growth and development and understanding how their disruption leads to spinal curvature remains the ultimate goal of this and similar investigations.