Japanese founder duplications/triplications involving BHLHA9 are associated with split-hand/foot malformation with or without long bone deficiency and Gollop-Wolfgang complex

Abstract

BackgroundLimb malformations are rare disorders with high genetic heterogeneity. Although multiple genes/loci have been identified in limb malformations, underlying genetic factors still remain to be determined in most patients.MethodsThis study consisted of 51 Japanese families with split-hand/foot malformation (SHFM), SHFM with long bone deficiency (SHFLD) usually affecting the tibia, or Gollop-Wolfgang complex (GWC) characterized by SHFM and femoral bifurcation. Genetic studies included genomewide array comparative genomic hybridization and exome sequencing, together with standard molecular analyses.ResultsWe identified duplications/triplications of a 210,050 bp segment containing BHLHA9 in 29 SHFM patients, 11 SHFLD patients, two GWC patients, and 22 clinically normal relatives from 27 of the 51 families examined, as well as in 2 of 1,000 Japanese controls. Families with SHFLD- and/or GWC-positive patients were more frequent in triplications than in duplications. The fusion point was identical in all the duplications/triplications and was associated with a 4 bp microhomology. There was no sequence homology around the two breakpoints, whereas rearrangement-associated motifs were abundant around one breakpoint. The rs3951819-D17S1174 haplotype patterns were variable on the duplicated/triplicated segments. No discernible genetic alteration specific to patients was detected within or around BHLHA9, in the known causative SHFM genes, or in the exome.ConclusionsThese results indicate that BHLHA9 overdosage constitutes the most frequent susceptibility factor, with a dosage effect, for a range of limb malformations at least in Japan. Notably, this is the first study revealing the underlying genetic factor for the development of GWC, and demonstrating the presence of triplications involving BHLHA9. It is inferred that a Japanese founder duplication was generated through a replication-based mechanism and underwent subsequent triplication and haplotype modification through recombination-based mechanisms, and that the duplications/triplications with various haplotypes were widely spread in Japan primarily via clinically normal carriers and identified via manifesting patients. Furthermore, genotype-phenotype analyses of patients reported in this study and the previous studies imply that clinical variability is ascribed to multiple factors including the size of duplications/triplications as a critical factor.Electronic supplementary materialThe online version of this article (doi:10.1186/s13023-014-0125-5) contains supplementary material, which is available to authorized users.

Femoral‐tibial‐digital malformations in a boy with the Japanese founder triplication of BHLHA9

“…We identified the Japanese founder triplication involving BHLHA9 in a boy with femoral‐tibial‐digital limb malformations. The results provide further support for the relevance of BHLHA9 overdosage in the occurrence of a range of limb malformations including femoral lesion [Nagata et al, ]. In this regard, the combination of metaphyseal widening, central metaphyseal radiolucency, and eccentric epiphyseal ossification center in the right distal femur appears to represent incomplete femoral bifurcation characteristic of GWC [Gollop et al, ; Wolfgang, ].…”

“…Recently, we have identified heterozygous tandem duplications/triplications of an identical 210,050 bp segment harboring BHLHA9 at chromosome band 17p13.3 in 27 of 51 Japanese families with split‐hand/foot malformation (SHFM), SHFM with long bone deficiency (SHFLD) that usually affects the tibia, or the Gollop‐Wolfgang complex (GWC) characterized by femoral bifurcation, tibial hypoplasia/aplasia, and SHFM [Nagata et al, ]. The results provide further support for the involvement of heterozygous copy number gains of BHLHA9 in the development of SHFM and SHFLD [Klopocki et al, ; Petit et al, ], and indicate for the first time the relevance of BHLHA9 overdosage in the occurrence of GWC.…”

“…These findings imply that the duplications/triplications involving BHLHA9 constitute a strong underlying factor with a dosage effect for the development of a range of limb malformations, with variable expressivity and incomplete penetrance. Furthermore, as the duplications/triplications have been generated between non‐homologous regions in association with a 4 bp microhomology at the fusion point, it is likely that a Japanese founder duplication was generated through a replication‐based mechanism and underwent subsequent triplication through recombination‐based mechanisms, and that the duplications/triplications were widely spread in Japan primarily via clinically normal carriers and identified via manifesting patients [Nagata et al, ]. Here, we report femoral‐tibial‐digital malformations in a Japanese boy with the founder triplication.…”

“…The results provide further support for the involvement of heterozygous copy number gains of BHLHA9 in the development of SHFM and SHFLD [Klopocki et al, ; Petit et al, ], and indicate for the first time the relevance of BHLHA9 overdosage in the occurrence of GWC. Notably, SHFLD and GWC are more frequent in families with triplications than in those with duplications, and the duplications/triplications are found not only in all the 42 affected patients, but also in 22 of 47 clinically normal relatives from the 27 families as well as in 2 of 1,000 Japanese controls [Nagata et al, ]. These findings imply that the duplications/triplications involving BHLHA9 constitute a strong underlying factor with a dosage effect for the development of a range of limb malformations, with variable expressivity and incomplete penetrance.…”

“…We performed genomewide array comparative genomic hybridization, and direct sequencing and quantitative real‐time PCR analysis for the fusion point of the Japanese founder duplication/triplication, after obtaining written informed consent and approval by the Institute Review Board Committee at Hamamatsu University School of Medicine. The methods were as reported previously [Nagata et al, ]. Consequently, we identified the Japanese founder triplication in the patient and the mother, but not in the father.…”