Elucidation of the phenotypic spectrum and genetic landscape in primary and secondary microcephaly

Abstract

Purpose: Microcephaly is a sign of many genetic conditions but has been rarely systematically evaluated. We therefore comprehensively studied the clinical and genetic landscape of an unselected cohort of patients with microcephaly.

Methods: We performed clinical assessment, high-resolution chromosomal microarray analysis, exome sequencing, and functional studies in 62 patients (58% with primary microcephaly [PM], 27% with secondary microcephaly [SM], and 15% of unknown onset).

Results: We found severity of developmental delay/intellectual disability correlating with severity of microcephaly in PM, but not SM. We detected causative variants in 48.4% of patients and found divergent inheritance and variant pattern for PM (mainly recessive and likely gene-disrupting [LGD]) versus SM (all dominant de novo and evenly LGD or missense). While centrosome-related pathways were solely identified in PM, transcriptional regulation was the most frequently affected pathway in both SM and PM. Unexpectedly, we found causative variants in different mitochondria-related genes accounting for ~5% of patients, which emphasizes their role even in syndromic PM. Additionally, we delineated novel candidate genes involved in centrosome-related pathway (SPAG5, TEDC1), Wnt signaling (VPS26A, ZNRF3), and RNA trafficking (DDX1).

Conclusion: Our findings enable improved evaluation and genetic counseling of PM and SM patients and further elucidate microcephaly pathways.

Keywords: MCPH; genetic counseling; mitochondria; primary microcephaly; secondary microcephaly.

Fig. 1

Exome sequencing (ES) coverage, growth parameters, and genetic evaluations of 62 patients with microcephaly. (a) Average coverages of targeted regions (left) and 20-fold average coverages (right) of ES data for all or mitochondrial genes. On average, ES yielded an average coverage of 222-fold (range: 92–419 fold) and covered about 96% of the targeted bases with ≥20 sequence reads and achieved an average off-target mitochondrial read depth of 43.6-fold (range: 3.9–163.9 fold) with a 20× average coverage of 64.4% (range: 1.9–99.4%). Distribution of average sequencing depth and 20× coverage of the targeted region was indistinguishable among patients with P/LP variants (red dots), high-level candidate variants (yellow dots), or others (VUS, [suspected] candidate, no candidate) (black dots). Mitochondrial genes exhibited significantly lower average coverages and 20-fold average coverages (Welch t test) with a higher variability in the 20-fold average coverages. P/LP pathogenic or likely pathogenic, VUS variant of uncertain significance. (b) SD distributions of growth parameters measured at birth and at the time of last investigation (variable ages). Connected lines represent individual cases. SDs below –2 (dotted line) were considered microcephaly. Dark green dots: primary microcephaly (PM, 36 [58.1%] patients); light green dots: secondary microcephaly (SM, 17 [27.4%] patients); gray dots: unknown onset (9 [14.5%] patients). Note that the distributions for OFC consistently show SD reductions at the last follow-up, suggesting progressiveness of microcephaly with a statistically significantly higher OFC reduction in PM compared with that in SM patients (p < 0.001, Wilcoxon rank-sum test). However, 61.3% of PM and 70.6% of SM patients did not show a decline in length or height similar to that in OFC, indicating a disproportionate microcephaly in the majority of our patients. OFC occipitofrontal head circumference, SD standard deviation (given as standard deviation score). (c) Distribution of (potentially) relevant genetic findings in the total cohort. Inner circle shows percentages of diagnostic and uncertain findings in established disease genes, as well as likely deleterious findings in candidate genes. Middle and outer circles show the distribution of CNVs and SVs, and the inheritance pattern in the respective categories of the inner circle, respectively. P/LP variants were identified in almost 50% of the patients. Most of these variants are SVs with comparable amounts of de novo (DN) occurrence and recessive inheritance. CNV copy-number variant, SV sequence variant. (d) Genetic findings in PM and SM. Diagnostic yields between PM (n = 36) and SM (n = 17) were comparable (left panel). Predominantly recessive inheritance was identified in diagnosed PM patients (~69%) and dominant de novo variants in all diagnosed SM patients (middle panel). Likely gene-disrupting (LGD) variants represented the most common disease alleles (~80%) among the diagnosed PM patients, while LGD and missense variants were equally observed among the diagnosed SM patients (right panel). CH compound heterozygous. Numbers on graphs were given as percentage.

Fig. 2

Facial photographs of selected patients with expanding clinical features or harboring high-level candidate genes. (a–d) Two phenotypically similar patients (ID32410 and 76870) with likely pathogenic variants in mitochondria-related genes MT-ATP6 and KARS at 15 years 3 months and 16 years 2 months, respectively. Note apparently closely spaced eyes, long nose with bulbous tip, apparently narrow mouth with crowded teeth, and large chin. (e–f) Patient 74812 with P/LP biallelic variants in PLK4, aborted at gestational week 23. Note sloping forehead, upslanting palpebral fissures, retrognathia, and apparently large ears with increased posterior angulation. (g–h) Patient 68629 with biallelic variants in a high-level candidate gene TEDC1 at 5 months (g) and 5 years 8 months (h). Note apparently broad forehead at young age, facial scoliosis (asymmetry with curvatures in relation to the vertical axis of the face), mild ptosis, beaked nose, apparently short ears, and micrognathia. (i–j) Patient 60361 with a de novo variant in a high-level candidate gene ZNRF3 at 4 years 9 months. Note sparse hair, left-sided microphthalmia with the secretions around both eyes due to lacrimal duct obstruction, narrow nose and nares, apparently large protruding ears, deep philtrum, thin lip vermilion (i), and oligodontia with conically shaped teeth (j). (k–l) Patient 74091 with homozygous variants in a high-level candidate gene DDX1 at 6 months. Note round face with mildly upslanting palpebral fissures, retrognathia, and apparently large ears with increased posterior angulation. P/LP pathogenic or likely pathogenic.

Fig. 3

Functional evaluations of high-level candidate variants in SPAG5 and TEDC1. (a) Determination of the allelic location of the de novo frameshift SPAG5 variant c.1223_1224insAC. A portion of SPAG5 sequence containing the frameshift variant and a nearby single-nucleotide polymorphism (SNP, rs113667723) was analyzed by Sanger sequencing of the patient’s blood DNA, which confirmed that the frameshift SPAG5 variant was located in the paternal allele by a distinct frameshift pattern of three bases around the SNP position. Blue sequence, paternal; pink sequence, maternal; black and underlined, variants. (b) Sanger sequencing of messenger RNA (mRNA) from the patient’s fibroblast (ID81652) showed a reduced amount of an aberrantly spliced transcript (due to the synonymous SPAG5 variant c.3189C>T with splice effect), which lacks the last 11 bp of exon 20, resulting in an out-of-frame mutation and a premature stop codon p.(Gly1064Glufs*3). In the magnified electropherogram of CHX, asterisk indicates rescued frameshift allele (nucleotide C in blue), leaky splice-site variant allele (nucleotide T in red), and rescued aberrantly spliced allele (nucleotide G in black). This means that the frameshift allele and the aberrantly spliced allele were rescued upon CHX treatment. CHX cycloheximide, DMSO dimethyl sulfoxide, WT wild type. (c) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed significantly reduced SPAG5 mRNA levels (~75%) in the patient’s fibroblasts (untreated and vehicle DMSO, p < 0.05, Welch t test), which were rescued upon treatment with CHX. Experiment was done in a triplicate. (d) Immunoblotting against the C-terminal terminal of SPAG5, detecting the two SPAG5 isoforms (full-length and short) and β-actin on protein extracts showed a significant reduction (~80%) of SPAG5 protein in the patient’s fibroblasts (ID81652) (p < 0.05, Welch t test). Note that the short isoform lacks a small portion of N-terminal of which the function has not yet been characterized. Experiment was done in a triplicate. (e) Immunostaining against SPAG5, PCNT, and α-Tubulin shows a reduced SPAG5 intensity mainly in the centrosomal regions where it is more condensed in the control during prophase to telophase. However, morphology of the patient’s fibroblasts appears with no obvious abnormality in the majority of cells (>95%). The nuclei were visualized by DAPI staining (in blue). The scale bar represents 10 μm. (f) RT-PCR showed higher expression levels of SPAG5 in normal human induced pluripotent stem cell–derived neural progenitor cells (NPCs) compared with fibroblasts and other cell types including testis (positive control), heart (negative control), HeLa cell line (highly proliferative control), and NPC-derived neuronal culture at 3 (NC3wks) or 5 (NC5wks) weeks. (g) Sanger sequencing of mRNA from the patient’s fibroblast (ID68629) showed a reduced amount of an aberrantly spliced transcript (due to the noncanonical splice-site TEDC1 variant c.227-5C>G that increases the activity of the cryptic splice acceptor), which lacks the first 40 bp of exon 3, resulting in an out-of-frame mutation and a premature stop codon p.(Glu76Glyfs*11). The levels of the aberrant transcript were rescued upon CHX treatment, indicating that the aberrant transcript was subjected to nonsense-mediated decay (NMD) (see also Figure S7). On the other hand, the sequencing of the other TEDC1 variant c.1111del, which is located in the last exon, did not show a reduced amount of the aberrant transcript. Nevertheless, this variant leads to a frameshift and premature stop codon p.(Ala371Glnfs*12) that removes the last 50 amino acids, likely leading to a deleterious effect on the function of the TEDC1 protein, which remains to be characterized. Bar graphs show the mean ± SEM.