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Background: Congenital microtia is a common craniofacial malformation resulting from both environmental and genetic factors. Recurrent chromosomal imbalances were observed in patients with microtia. The 22q11.2 deletion is one of the most common microdeletions in human beings. The cell division cycle 45 gene (CDC45) embedded in the proximal 22q11.2 deleted region is involved in craniofacial development. However, only a few studies have focused on the 22q11.2 deletion as genetic etiology in microtia patients and studied its associated external ear deformity characteristics in detail. Methods: In this research, a total of 65 patients from north China with sporadic microtia were studied. Copy number variations of CDC45 were screened using AccuCopy assay. The 22q11.2 deletion harboring CDC45 was identified by whole‐genome sequencing and targeted next‐generation sequencing. A parental test was carried out to determine the origin of the deletion. Results: CDC45 copy number loss was identified in two patients with microtia. A set of qPCR assays demonstrated two patients carried a typical proximal 22q11.2 deletion between the low‐copy repeats on chromosome 22q11.2 (LCR22A and LCR22D), encompassing CDC45. The 22q11.2 deletions were de novo in each patient. In‐depth auricular phenotype assessment showed these two patients have a distinct concha‐type ear malformation while other microtia patients have lobule‐type microtia among the 65 microtia patient cohort in this study. Conclusion: Here we present two additional Chinese microtia patients with de novo 22q11.2 proximal deletion harboring CDC45 and further report these patients' distinct ear malformation. [ABSTRACT FROM AUTHOR]

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 malformations, as in other species. This study also provides an additional example of functional conserved non-coding elements (CNEs) in humans. [ABSTRACT FROM AUTHOR]

• Two bilateral non-syndromic microtia cases with HOXA2 mutations of Chinese origin were reported. • A novel nonsense HOXA2 mutation was identified, and a mutational hotspot is implicated. • These mutations could impair the activation of HMX1 by interacting with a long-range enhancer. HOX genes are important regulatory genes patterning head formation, including development of the ear. Microtia is a congenital ear anomaly characterized by lacking all or part of the structures of the outer ear. To date, only four HOXA2 mutations were reported in families with autosomal-recessive or dominant microtia, with or without hearing impairment. More identified mutations are needed to confirm the correlation between genotype and phenotype. Here, we collect two Chinese families with non-syndromic bilateral microtia. Next generation sequencing identified two heterozygous nonsense HOXA 2 mutations, one in each family. One mutation (c.637A > T, p.Lys213*) is newly reported, while the other one (c.703C > T,p.Gln235*) is consistent with a previous report. In mouse, Hoxa2 can bind to a long-range enhancer and regulate expression of the Hmx1 gene, which is a crucial transcription factor in eye and ear development. Using dual luciferase reporter assays, we showed that both HOXA2 mutations have impaired activation of the long-range enhancer of HMX1. In the present study, we report the first two bilateral non-syndromic microtia cases with HOXA2 mutations of Chinese origin and identify a novel mutation. Our results also provide molecular insights about how these nonsense HOXA2 mutations could affect the activation of its downstream target HMX1 by interacting with the long-range enhancer. [ABSTRACT FROM AUTHOR]

• Novel KCTD1 mutation (NM_198991, c.G185T, p.G62V) in Chinese patient. • Characteristic facial features including hypertelorism, epicanthus, flat nose, and prominent thick subcutaneous fat layer between eyes. • KCTD1 G62V mutant impairs the activity of the AP-2α and Wnt/β-catenin pathway. Microtia is a common birth defect affecting the external ears and encompasses a spectrum of congenital anomalies of the auricle. For some of the microtia-associated syndromes, the additional abnormalities are not easily observed or with variable expressivity. Identifying pathogenic mutations through genetic testing is of great help in recognizing these highly heterogeneous syndromes in clinical practice. We reported a novel de novo KCTD1 mutation in a Chinese patient with congenital microtia. It expands the mutational spectrum of KCTD1 and provide an additional scalp‐ear‐nipple patient with typical and atypical clinical presentations. The identified mutation in the BTB domain impairs the suppressive activity of the AP-2 transcription factor family and may impact on maintaining the finely tuned activity of WNT pathway, which directs stem cell development in ectoderm patterning and craniofacial development. Due to the variable expressive clinical phenotypes of syndromic microtia, genetic molecular testing could be of great help in the definite diagnosis. [ABSTRACT FROM AUTHOR]