Clinical spectrum of females with HCCS mutation: from no clinical signs to a neonatal lethal form of the microphthalmia with linear skin defects (MLS) syndrome

Abstract

Background: Segmental Xp22.2 monosomy or a heterozygous HCCS mutation is associated with the microphthalmia with linear skin defects (MLS) or MIDAS (microphthalmia, dermal aplasia, and sclerocornea) syndrome, an X-linked disorder with male lethality. HCCS encodes the holocytochrome c-type synthase involved in mitochondrial oxidative phosphorylation (OXPHOS) and programmed cell death.

Methods: We characterized the X-chromosomal abnormality encompassing HCCS or an intragenic mutation in this gene in six new female patients with an MLS phenotype by cytogenetic analysis, fluorescence in situ hybridization, sequencing, and quantitative real-time PCR. The X chromosome inactivation (XCI) pattern was determined and clinical data of the patients were reviewed.

Results: Two terminal Xp deletions of ≥ 11.2 Mb, two submicroscopic copy number losses, one of ~850 kb and one of ≥ 3 Mb, all covering HCCS, 1 nonsense, and one mosaic 2-bp deletion in HCCS are reported. All females had a completely (>98:2) or slightly skewed (82:18) XCI pattern. The most consistent clinical features were microphthalmia/anophthalmia and sclerocornea/corneal opacity in all patients and congenital linear skin defects in 4/6. Additional manifestations included various ocular anomalies, cardiac defects, brain imaging abnormalities, microcephaly, postnatal growth retardation, and facial dysmorphism. However, no obvious clinical sign was observed in three female carriers who were relatives of one patient.

Conclusion: Our findings showed a wide phenotypic spectrum ranging from asymptomatic females with an HCCS mutation to patients with a neonatal lethal MLS form. Somatic mosaicism and the different ability of embryonic cells to cope with an OXPHOS defect and/or enhanced cell death upon HCCS deficiency likely underlie the great variability in phenotypes.

Figure 1

Pedigree, FISH and X chromosome inactivation of patient 1 and three healthy female relatives. A. Pedigree of patient’s 1 family. Patient 1 (III-1) is affected by MLS syndrome, while her mother (II-1), her maternal aunt (II-2) and her maternal grandmother (I-1) are asymptomatic. FISH analysis with BAC RP11-163I1, spanning the HCCS gene on metaphase spreads of the four females revealed one signal in patient 1 (III-1), her mother (II-1) and her aunt (II-2). For the maternal grandmother (I-1), two signals for RP11-163I1 and three signals for the X centromere probe DXZ1 were obtained. BAC RP11-163I1 and DXZ1 were labelled with Spectrum Green-dUTP. Arrows point to the wild-type X chromosome (WT X) and the X chromosome with the microdeletion at Xp22.2 (del(X)). X chromosome inactivation was determined by analysing the methylation status of the androgen receptor gene at Xq12. Predigestion of genomic DNA isolated from lymphocytes with and without HpaII is indicated by (+) and (-), respectively. Representative electropherograms show the different AR alleles (designated as 1, 2, 3 and 4) in the four females. Females II-1, II-2 and III-1 have extremely skewed X inactivation (upper electropherograms indicated with +). B. Physical map of part of the Xp22.3 and Xp22.2 regions that are indicated by horizontal grey bars; Mbs and the telomere (tel) to centromere (cen) orientation are given. Arrows represent selected genes in Xp22 and gene symbols are given; arrowheads indicate the 5′ → 3′ transcription direction of the genes. BAC RP11-163I1 (RP11 Human BAC Library) and four Xp22.2 fosmid (WIBR-2 Human Fosmid Library) clones are indicated by black bars and names are given. Interstitial deletions found in patients 1 (P1) and 6 (P6) are depicted as horizontal black lines and the size of each deletion is given. Dotted and wavy lines indicate that the deletion breakpoints were not fine-mapped.

Figure 2

Copy number analysis of HCCS and neighboring genes by quantitative real-time PCR in patient 6. A. Relative quantification of copy number of HCCS exons 1, 3, 6 and 7 by qPCR on genomic DNA of patient 6 (black bars) and her mother (darkgrey bars) revealed values that are comparable to a haploid sample (lightgrey bars) and a diploid sample (white bars), respectively. White, lightgrey and black bars represent the mean ± SD of two independent experiments, each performed in duplicate. The darkgrey bars represent the mean of one experiment performed in duplicate for each exon. B. Relative quantification of copy number of HCCS surrounding genes in patient 6. qPCR for KAL1 exon 8 and RAB9A exon 3 on genomic DNA of patient 6 (black bars) revealed values that were comparable to a diploid sample (white bars), while those for SHROOM2 exon 6 and TLR7 exon 3 (black bars) were comparable with a haploid sample (lightgrey bars). Each bar represents the mean ± SD of at least two experiments performed in duplicate.

Figure 3

HCCS sequence-level mutations in patients 2 and 3. A. Sequence electropherogram from genomic DNA of patient 2 showing part of HCCS exon 6. Nucleotide triplets and encoded amino acids (one letter code) are indicated above the electropherogram. The red arrow points to the double peak in the electropherogram showing heterozygosity for the nonsense mutation c.589C > T (p.R197*). B. Sequence electropherograms of part of HCCS exon 6 from DNA isolated from leukocytes (top), buccal cells (middle) and the lymphoblastoid cell line (LCL; bottom) of patient 3. Nucleotide triplets and encoded amino acids (one letter code) are shown for the wild-type and mutant allele above the electropherograms. The red arrow points to the first double peak in the electropherogram indicating the start of the frameshift. In LCL-derived DNA only HCCS wild-type sequence was visible in the electropherogram. Patient 3 carries the mosaic frameshift mutation c.[=/524_525delAG] / p.[=/E175Vfs*30] in HCCS.

Figure 4

Photographs of the six patients with MLS syndrome and a heterozygous HCCS mutation or Xp22 monosomy. A and B. Patient 2 died at the age of 4 months. She presented with microphthalmia and sclerocornea of the right eye and anophthalmia of the left eye (A). Linear skin defects were observed on her neck (B). C. In the 7-year-old patient 5, linear skin defects and small hemangiomas were noted. She showed microphthalmia and sclerocornea of the left eye and anophthalmia of the right eye. D. Microphthalmia and sclerocornea of both eyes were present in patient 1, while linear skin defects were absent. E. In patient 3 (age 11 months), microphthalmia and sclerocornea of the left eye were diagnosed. No linear skin defects were noted. F, G and H. Linear skin defects on the face of patient 4 were very prominent at birth (F, 4 days old), but healed with age (3 weeks old in G and 2 years old in H). Severe bilateral microphthalmia was observed in the patient. I. Bilateral microphthalmia and sclerocornea were observed in patient 6; she also had typical linear skin defects on her face (photographs submitted with written consent from the patients’ legal guardians for publication in print and online).