Microcephaly, intellectual impairment, bilateral vesicoureteral reflux, distichiasis, and glomuvenous malformations associated with a 16q24.3 contiguous gene deletion and a Glomulin mutation

Abstract

Two hereditary syndromes, lymphedema-distichiasis (LD) syndrome and blepharo-chelio-dontic (BCD) syndrome include the aberrant growth of eyelashes from the meibomian glands, known as distichiasis. LD is an autosomal dominant syndrome primarily characterized by distichiasis and the onset of lymphedema usually during puberty. Mutations in the forkhead transcription factor FOXC2 are the only known cause of LD. BCD syndrome consists of autosomal dominant abnormalities of the eyelid, lip, and teeth, and the etiology remains unknown. In this report, we describe a proband that presented with distichiasis, microcephaly, bilateral grade IV vesicoureteral reflux requiring ureteral re-implantation, mild intellectual impairment and apparent glomuvenous malformations (GVM). Distichiasis was present in three generations of the proband's maternal side of the family. The GVMs were severe in the proband, and maternal family members exhibited lower extremity varicosities of variable degree. A GLMN (glomulin) gene mutation was identified in the proband that accounts for the observed GVMs; no other family member could be tested. TIE2 sequencing revealed no mutations. In the proband, an additional submicroscopic 265 kb contiguous gene deletion was identified in 16q24.3, located 609 kb distal to the FOXC2 locus, which was inherited from the proband's mother. The deletion includes the C16ORF95, FBXO31, MAP1LC3B, and ZCCHC14 loci and 115 kb of a gene desert distal to FOXC2 and FOXL1. Thus, it is likely that the microcephaly, distichiasis, vesicoureteral, and intellectual impairment in this family may be caused by the deletion of one or more of these genes and/or deletion of distant cis-regulatory elements of FOXC2 expression.

Figure 1. Transmission of the Phenotypes in the Family of the Proband

Three generations of the proband’s family are shown by pedigree. Each generation has affected individuals indicating possible autosomal dominant inheritance of distichiasis and vascular malformations, but with variable expressivity for vascular malformations. Symbols used in the pedigree are shown in the legend.

Figure 2. Vascular Malformations

VMs of right lateral brow (A), v-shaped reticular lesions on the left side of the chest extending from the scapula to below the sternum (B), lesions along the left arm (C), vascular malformations of the posterior lower left leg at the level of knee (D), vascular malformations of the left ankle and plantar surface of the foot (E), and lesions of the left second finger (F). Prominent superficial vasculature is exhibited in the left leg of the maternal grandmother of the proband (G).

Figure 3. MRI Imaging of the Vascular Anomalies of the Proband

An infiltrative lesion (large arrows) is seen mainly within the soleus and flexor hallucis longus muscles, sparing the overlying distal medial gastrocnemius muscle. On T1 weighting (A), the intramuscular lesion is subtle and similar in signal intensity to the adjacent normal musculature. Scattered tiny areas of high signal may represent minimal fat within the lesion. On fluid sensitive sequences (B & D), the lesion is of high signal. After gadolinium is administered (C), there is mild enhancement within the intramuscular lesion which increases on later images (E). Best seen on the coronal images (D and E), the lesion appears composed of small vascular-like channels. The lesion extends into the adjacent subcutaneous fat (A–D, small arrows). The subcutaneous component also has the appearance of vascular-like channels. The overall appearance is most consistent with a vascular lesion, likely a venous malformation. The abnormality extends to the ankle (D, double arrows).

Figure 4. FOXC2 Deletion Screening

A. Diagram of the genomic insert of BAC clone RP11-463O9 including the genes (gray boxes) and intergenic distances (kilobases). Exon/intron structure of MTHFSD is not shown for simplicity. B. FISH using RP11-463O9 as probe generated hybridization signals on both chromosome 16 homologs (white arrows) of the proband.

Figure 5. Mapping of the 16q24 Contiguous Gene Deletion

A. Physical map of the 16q24 region showing the proximity of the deletion with respect to FOXC2. Genes are depicted by gray boxes ignoring exon/intron structure. The dashed lines show the relative position of a 612 kb segment extending from BAC clone RP11-235F18 to RP11-482M8. Genes in this magnified region are represented by arrows showing the direction of transcription for each gene. BAC clones used for FISH are shown as gray (1 signal-deleted) and black (2 signals-present) lines. SNPs rs1465452 and rs4843612 SNPs are marked by vertical black lines. B–D. FISH on metaphase chromosomes from the proband. FISH was performed with BAC clone RP11-235F18 (B), RP11-178L8 (C), and RP11-482M8 (D). In panel C, RP11-264L1 (red) was co-hybridized with RP11-178L8 (green) as a control probe to mark each chromosome 16. White arrows mark hybridization signals. The red arrow marks the chromosome 16 carrying the deletion demonstrated by the absence of a green (RP11-178L8) hybridization signal (panel C).

Figure 6. Cloning of the 16q24 Deletion by Inverse PCR

A. 16q24.3 deletion breakpoints. The genomic sequence surrounding the proximal and distal breakpoints of the 16q24.3 deletion is shown above a sequencing chromatogram from the pMTB3-1-2 inverse PCR clone (deleted sequence is shaded). UCSC genome browser coordinates are shown above the sequence. Breakpoints are indicated by red lines. Deleted sequence is shown in lowercase whereas present sequence is shown in uppercase. B. Confirmation of 16q24 deletion breakpoints. A common forward primer (*) proximal to the 16q24 deletion in combination with reverse primers specific for the wild type allele (black) and the deletion allele (red) were used for PCR. The proband (P) was heterozygous for the 16q24 deletion whereas an unrelated control (C) was homozygous for the wild type allele. A control PCR reaction with no template was also performed (H). C. The 16q24 deletion removes C16ORF95, FBXO31, MAP1LC3B, and ZCCHC14 loci. The 16q24 region is shown as a black horizontal line with vertical lines representing exons and arrows showing the direction of the corresponding transcripts. The 16q24 deletion is depicted below by a red line.