Duplications involving the long range HMX1 enhancer are associated with human isolated bilateral concha-type microtia

Abstract

Background: Microtia is a congenital anomaly of ear that ranges in severity from mild structural abnormalities to complete absence of the outer ears. Concha-type microtia is considered to be a mild form. The H6 family homeobox 1 transcription factor gene (HMX1) plays an important role in craniofacial structures development. Copy number variations (CNVs) of a downstream evolutionarily conserved enhancer region (ECR) of Hmx1 associated with ear and eye abnormalities have been reported in different animals, but not yet in human. To date, no genetic defects responsible for isolated human microtia has been reported except for mutations in HOXA2. Here we recruited five Chinese families with isolated bilateral concha-type microtia, and attempt to identify the underlying genetic causes. Methods: Single Nucleotide polymorphism (SNP) array was performed to map the disease locus and detect CNVs on a genome scale primarily in the largest family (F1). Whole genome sequencing was performed to screen all SNVs and CNVs in the candidate disease locus. Array comparative genomic hybridization (aCGH) was then performed to detect CNVs in the other four families, F2-F5. Quantitative real-time polymerase chain reaction (qPCR) was used to validate and determine the extent of identified CNVs containing HMX1-ECR region. Precise breakpoints in F1 and F2 were identified by gap-PCR and sanger sequencing. Dual-luciferase assays were used to detect the enhancer function. qPCR assays were also used to detect HMX1-ECR CNVs in 61 patients with other types mictrotia. Results: Linkage and haplotype analysis in F1 mapped the disease locus to a 1.9 Mb interval on 4p16.1 containing HMX1 and its downstream ECR region. Whole genome sequencing detected no potential pathogenic SNVs in coding regions of HMX1 or other genes within the candidate disease locus, but it detected a 94.6 Kb duplication in an intergenic region between HMX1 and CPZ. aCGH and qPCRs also revealed co-segregated duplications in intergenic region downstream of HMX1 in the other four families. The 21.8 Kb minimal overlapping region encompassing the core sequences consensus with mouse ECR of Hmx1. Luciferase assays confirmed the enhancer function in human sequences, and proved that HOXA2 could increase its enhancer activity. No CNVs were detected in HMX1-ECR regions in 61 patients with other type of microtia. Conclusion: Duplications involving long range HMX1 enhancers are associated with human isolated bilateral concha-type microtia. We add to evidences in human that copy number variations in HMX1-ECR associates with ear

Oculo-auriculo-vertebral spectrum: new genes and literature review on a complex disease

“…Duplications at 4p16.1 including HMX1 were also associated with OAVS 44 45. Of note, this duplication probably excludes the HMX1 downstream evolutionarily conserved enhancer region whose duplication is associated with bilateral concha-type microtia 46. Among other factors, HOXA2 is able to link this enhancer and interestingly, HOXA2 loss-of-function variants are also associated with bilateral microtia [MIM:612 290],47 48 whereas HMX1 biallelic loss-of-function variants are associated with oculoauricular syndrome [MIM:612 109] 49–51.…”

“…This specific non-coding genomic region was previously implicated in external ear development in vertebrates 87. Interestingly, HMX1 biallelic variants are associated with oculoauricular syndrome [MIM:612 109]51 whereas the specific duplication of HMX1 -ECR is linked to Concha-type microtia, an autosomal-dominant mild form of microtia 46. Several spontaneous animal models of microtia/ear dysplasia (cattle or sheep) are described as carrying duplications of the HMX1 -ECR88–90 (table 4).…”

OTX2duplications: a recurrent cause of oculo-auriculo-vertebral spectrum

“…The 22q11.2 locus is regularly associated with OAVS, without clear pathophysiological mechanisms to explain it so far 23. 4p16 duplications involving HMX1 or his long-range enhancer were reported in OAVS individuals 24–26. Lastly, large 14q22.3 duplications were also associated with OAVS in three previous studies,27–29 and OAVS clinical features were described in a patient with an inversion breakpoint in 14q22.3 30.…”

Genetic basis of ear length in sheep breeds sampled across the region from the Middle East to the Alps

“…Moreover, this study suggested a significant association and interaction between the duplication in the ECR near to HMX1 (OAR6) and the missense mutation in exon 7 of GATA6 (OAR23). Interestingly, a study (Si et al., 2020) reported that a duplication in the enhancer region of the HMX1 locus is associated with a type of microtia in humans. Furthermore, a study of this trait in Highland Cattle, utilising SNP‐chip genotyping and sequencing‐based fine mapping, also yielded HMX1 as a candidate locus (Koch et al., 2013).…”