17p13.3 genomic rearrangement in a Chinese family with split-hand/foot malformation with long bone deficiency: report of a complicated duplication with marked variation in phenotype

Abstract

Background: Split hand/foot malformation (SHFM) is a genetically heterogeneous limb malformation with variable expressivity. SHFM with tibia or femur aplasia is called SHFM with long bone deficiency (SHFLD). 17p13.3 duplications containing BHLHA9 are associated with SHFLD. Cases with variable SHFLD phenotype and different 17p13.3 duplicated regions are reported. The severity of long bone defect could not be simply explained by BHLHA9 overdosage or 17p13.3 duplication.

Methods: A four-generation Chinese SHFM family was recruited. Three family members have long bone defects, one male was severely affected with hypoplasia or aplasia in three of four limbs. Linkage analysis and direct sequencing of candidate genes were used to exclude six responsible genes/loci for isolated SHFM. Array comparative genomic hybridization (CGH) was performed to detect copy number variations on a genome-wide scale, and quantitative real-time polymerase chain reaction (qPCR) assays were designed to validate the identified copy number variation in the index and other family members.

Results: No mutations were found in genes or loci linked to isolated SHFM. A ~ 966 kb duplication was identified in 17p13.3 by array CGH, in which BHLHA9 surrounding region presented as triplication. The qPCR assays confirmed the indicated 17p13.3 duplication as well as BHLHA9 triplication in all available affected family members and other two asymptomatic carriers. Given the incomplete penetrance in SHFLD, those two carriers were regarded as non-penetrant, which suggested that the genomic rearrangement was co-segregated with malformation in this family.

Conclusions: The present study reports an additional SHFLD family case with 17p13.3 genomic rearrangement. To our knowledge, the 966 kb genomic rearrangement is larger in size than any previously reported SHFLD-associated 17p13.3 duplication, and the present family shows marked phenotypic variability with two asymptomatic carriers and one patient with an extremely severe phenotype. This rare case provides the opportunity to identify underlying genotype-phenotype correlations between SHFLD and 17p13.3 genomic rearrangement.

Keywords: 17p13.3 genomic rearrangement; BHLHA9; Split hand/foot malformation with long bone deficiency.

Fig. 1

Pedigree and limb defects of affected subjects. a Pedigree showing dominant inheritance with reduced penetrance. b Ectrodactyly with split hand of patient I1. c-d Ectrodactyly with split left hand of II1 (c). The hypoplastic right forearm with no hand, ulna and radius of II1 (d). Patient II1 also affected with symmetric hypoplastic femora and absence of bilateral lower legs. e Unilateral ectrodactyly with split hand of II6. f. Unilateral clinodactyly in middle digit of II8. g Abnormal left split hand of the aborted fetus IV6

Fig. 2

Identification and validation of 17p13.3 genomic rearrangement. a Array CGH results (GRCh37/hg19 build) showed copy number gain at the chromosome 17p13.3 locus. A large duplication was identified here and part of this region encompassing ABR1 and BHLHA9 manifested as triplication. The blue and the red dots denote amplification and deletion, respectively. The log ratio (Y axis) demonstrated by the blue dots indicated the presence of increased copies of this region. Ratios around + 0.5 and around + 1.0 indicate the presence of three and four copies of its corresponding region, respectively. b DNA copy numbers of 17p13.3 genomic rearrangement region detected by qPCR assays. Primers locations are indicated at the bottom of a (black bars). c DNA copy number of BHLHA9 detected by qPCR assays. All the normal subjects except III6 and III8 in this family show normal copy number (white bars). All the affected individuals show increased copy numbers (black bars). C1 and C2 are two normal non-familial individuals, which represent a male and a female respectively

Fig. 3

Schematic overview of the 17p13.3 duplications previously reported and identified in our study (hg19). All the twenty-two blue blocks denote the previously reported duplicated regions. The lower red block denotes the identified duplication and the grey block denotes the identified triplication in our study. The complicated duplication found in our study contained a wider telocentric region than in previously reported 17p13.3 duplications