Abstract：
Split-hand/ foot malformation with long bone deficiency 3 (SHFLD3) is an extremely rare condition associated with duplications located on 17p13.3, which invariably encompasses the BHLHA9 gene. The disease inherits with variable expressivity and significant incomplete penetrance as high as 50%.
We have detected 17p13.3 locus one-allele triplication in a male proband from family 1 (F1.1), and duplication in a male proband from family 2 (F2.1) applying array comparative genomic hybridization (array CGH). The rearrangements mapped to the following chromosomal regions-arr[GRCh38] 17p13.3(960254-1291856)×4 in F1.1 and arr[GRCh38] 17p13.3(1227482-1302716)×3 in F2.1. The targeted quantitative PCR revealed that the 17p13.3 locus was also duplicated in the second affected member from family 2 (F2.2; brother of F2.1). In the next step, we performed segregation studies using quantitative PCR and revealed that F1.1 inherited the triplication from his healthy father-F1.2, whereas the locus was unremarkable in the mother of F2.1 & F2.2 and the healthy son of F2.1. However, the duplication was present in a healthy daughter of F2.2, an asymptomatic carrier. The breakpoint analysis allowed to define the exact size and span of the duplicated region in Family 2, i.e., 78,948 bp chr17:1225063-1304010 (HG38). Interestingly, all symptomatic carriers from both families presented with variable SHFLD3 phenotype. The involvement of secondary modifying locus could not be excluded, however, the Sanger sequencing screening of BHLHA9 entire coding sequence was unremarkable for both families.
We have shed light on the one-allele CNV triplication occurrence that should be considered when a higher probe (over duplication range) signal is noted. Second, all SHFLD3 patients were accurately described regarding infrequent limb phenotypes, which were highly variable even when familial. Of note, all symptomatic individuals were males. SHFLD3 still remains a mysterious ultra-rare disease and our findings do not answer crucial questions regarding the disease low penetrance, variable expression and heterogeneity. However, we have presented some clinical and molecular aspects that may be helpful in daily diagnostic routine, both dysmorphological and molecular assessment, of patients affected with SHFLD3.
We have detected 17p13.3 locus one-allele triplication in a male proband from family 1 (F1.1), and duplication in a male proband from family 2 (F2.1) applying array comparative genomic hybridization (array CGH). The rearrangements mapped to the following chromosomal regions-arr[GRCh38] 17p13.3(960254-1291856)×4 in F1.1 and arr[GRCh38] 17p13.3(1227482-1302716)×3 in F2.1. The targeted quantitative PCR revealed that the 17p13.3 locus was also duplicated in the second affected member from family 2 (F2.2; brother of F2.1). In the next step, we performed segregation studies using quantitative PCR and revealed that F1.1 inherited the triplication from his healthy father-F1.2, whereas the locus was unremarkable in the mother of F2.1 & F2.2 and the healthy son of F2.1. However, the duplication was present in a healthy daughter of F2.2, an asymptomatic carrier. The breakpoint analysis allowed to define the exact size and span of the duplicated region in Family 2, i.e., 78,948 bp chr17:1225063-1304010 (HG38). Interestingly, all symptomatic carriers from both families presented with variable SHFLD3 phenotype. The involvement of secondary modifying locus could not be excluded, however, the Sanger sequencing screening of BHLHA9 entire coding sequence was unremarkable for both families.
We have shed light on the one-allele CNV triplication occurrence that should be considered when a higher probe (over duplication range) signal is noted. Second, all SHFLD3 patients were accurately described regarding infrequent limb phenotypes, which were highly variable even when familial. Of note, all symptomatic individuals were males. SHFLD3 still remains a mysterious ultra-rare disease and our findings do not answer crucial questions regarding the disease low penetrance, variable expression and heterogeneity. However, we have presented some clinical and molecular aspects that may be helpful in daily diagnostic routine, both dysmorphological and molecular assessment, of patients affected with SHFLD3.
摘要：
具有长骨缺陷3（SHFLD3）的手/足畸形是一种极为罕见的疾病，与位于17p13.3上的重复有关，该重复总是包含BHLHA9基因。该疾病具有可变的表现力和高达50％的显着不完全外显率。
我们在家族1（F1.1）的男性先证者中检测到17p13.3基因座单等位基因三重复，以及应用阵列比较基因组杂交（阵列CGH）在来自家族2（F2.1）的男性先证者中的重复。重排映射到以下染色体区域-F1.1中的arr[GRCh38]17p13.3（960254-1291856）×4和F2.1中的arr[GRCh38]17p13.3（1227482-1302716）×3。靶向定量PCR显示17p13.3基因座也在家族2的第二个受影响成员中重复(F2.2;F2.1的兄弟)。下一步,我们使用定量PCR进行了分离研究，发现F1.1继承了他健康父亲F1.2的三倍体，而F2.1和F2.2的母亲和F2.1的健康儿子的基因座并不明显。然而,重复存在于F2.2的健康女儿中,F2.2是无症状携带者.断点分析允许定义Family2中重复区域的确切大小和跨度，即78,948bpchr17：1225063-1304010（HG38）。有趣的是,来自两个家庭的所有有症状的携带者均呈现可变的SHFLD3表型。不能排除二级修饰位点的参与，然而,BHLHA9完整编码序列的Sanger测序筛选在两个家族中都不显著.
我们已经阐明了当注意到更高的探针(超过复制范围)信号时应该考虑的单等位基因CNV三重复发生。第二,所有SHFLD3患者都被准确描述为不常见的肢体表型，即使是家族性的，也是高度可变的。值得注意的是，所有有症状的个体均为男性.SHFLD3仍然是一种神秘的超罕见疾病，我们的发现并没有回答关于疾病低外显率的关键问题。变量表达和异质性。然而,我们已经提出了一些临床和分子方面，可能有助于日常诊断，形态学和分子评估，患有SHFLD3的患者。
我们在家族1（F1.1）的男性先证者中检测到17p13.3基因座单等位基因三重复，以及应用阵列比较基因组杂交（阵列CGH）在来自家族2（F2.1）的男性先证者中的重复。重排映射到以下染色体区域-F1.1中的arr[GRCh38]17p13.3（960254-1291856）×4和F2.1中的arr[GRCh38]17p13.3（1227482-1302716）×3。靶向定量PCR显示17p13.3基因座也在家族2的第二个受影响成员中重复(F2.2;F2.1的兄弟)。下一步,我们使用定量PCR进行了分离研究，发现F1.1继承了他健康父亲F1.2的三倍体，而F2.1和F2.2的母亲和F2.1的健康儿子的基因座并不明显。然而,重复存在于F2.2的健康女儿中,F2.2是无症状携带者.断点分析允许定义Family2中重复区域的确切大小和跨度，即78,948bpchr17：1225063-1304010（HG38）。有趣的是,来自两个家庭的所有有症状的携带者均呈现可变的SHFLD3表型。不能排除二级修饰位点的参与，然而,BHLHA9完整编码序列的Sanger测序筛选在两个家族中都不显著.
我们已经阐明了当注意到更高的探针(超过复制范围)信号时应该考虑的单等位基因CNV三重复发生。第二,所有SHFLD3患者都被准确描述为不常见的肢体表型，即使是家族性的，也是高度可变的。值得注意的是，所有有症状的个体均为男性.SHFLD3仍然是一种神秘的超罕见疾病，我们的发现并没有回答关于疾病低外显率的关键问题。变量表达和异质性。然而,我们已经提出了一些临床和分子方面，可能有助于日常诊断，形态学和分子评估，患有SHFLD3的患者。
