Neurogenetic fetal akinesia and arthrogryposis: genetics, expanding genotype-phenotypes and functional genomics

Abstract

Background: Fetal akinesia and arthrogryposis are clinically and genetically heterogeneous and have traditionally been refractive to genetic diagnosis. The widespread availability of affordable genome-wide sequencing has facilitated accurate genetic diagnosis and gene discovery in these conditions.

Methods: We performed next generation sequencing (NGS) in 190 probands with a diagnosis of arthrogryposis multiplex congenita, distal arthrogryposis, fetal akinesia deformation sequence or multiple pterygium syndrome. This sequencing was a combination of bespoke neurogenetic disease gene panels and whole exome sequencing. Only class 4 and 5 variants were reported, except for two cases where the identified variants of unknown significance (VUS) are most likely to be causative for the observed phenotype. Co-segregation studies and confirmation of variants identified by NGS were performed where possible. Functional genomics was performed as required.

Results: Of the 190 probands, 81 received an accurate genetic diagnosis. All except two of these cases harboured class 4 and/or 5 variants based on the American College of Medical Genetics and Genomics guidelines. We identified phenotypic expansions associated with CACNA1S, CHRNB1, GMPPB and STAC3. We describe a total of 50 novel variants, including a novel missense variant in the recently identified gene for arthrogryposis with brain malformations-SMPD4.

Conclusions: Comprehensive gene panels give a diagnosis for a substantial proportion (42%) of fetal akinesia and arthrogryposis cases, even in an unselected cohort. Recently identified genes account for a relatively large proportion, 32%, of the diagnoses. Diagnostic-research collaboration was critical to the diagnosis and variant interpretation in many cases, facilitated genotype-phenotype expansions and reclassified VUS through functional genomics.

Keywords: clinical genetics; molecular genetics; neuromuscular disease.

Figure 1:. Bi-allelic class 3 variants in CACNA1S and SCN4A associated with fetal akinesia.

(A) A pedigree co-segregating bi-allelic missense variants in CACNA1S with fetal akinesia and (B) conservation across species at the observed substitutions: p.Met222 and p.Arg789. (C) Evolutionarily conservation at two SCN4A residues substituted, bi-allelically, in another case of fetal akinesia. Importantly, previous work substituting p.Ser1478 to cysteine showed enhanced inactivation of the Na⁺ channel. Abbreviations: d. 10 d = died at 10 days of age, TOP 26wg = termination of pregnancy at 26 weeks gestation.

Figure 2:. Homozygous deletion within CHRNB1 causes lethal multiple pterygia syndrome.

(A) A nonconsanguineous family with recurrent lethal multiple pterygia syndrome; IUFD: in utero fetal demise and TOP: termination of pregnancy . (B) Visualisation of the CHRNB1 exon 8 deletion in the exome sequencing data from the proband (blue trace) and both parents (purple traces). The grey lines represent other samples in the same batch of CNV calling and show the average amount of noise across the region. The y-axis denotes the copy number as inferred by gCNV, and the x-axis shows the position along the gene in kilobases. The dots on the plot represent a probe for exon capture, which roughly represent exons. The copy number estimation is expected to hover around 2.0 for autosomes. For the X chromosome, the copy number estimation should hover around 2.0 for females and 1.0 for males.

Figure 3:. Clinical presentation in a case with mild distal arthrogryposis due to a de novo missense variant in FLNC.

Images A and B were taken at age 8 months and image C was taken at age 2 months. The images demonstrate reduced elbow extension with dimples, excessive ankle hypermobility, and subtle facial findings including plagiocephaly and micrognathia.

Figure 4:. Homozygous variant in STAC3.

(A) Pedigree showing segregation of a homozygous variant in STAC3. (B) 2% agarose gel of RT-PCR products from the STAC3 minigene assay. WT – normal exon 3 of STAC3, MUT – exon 3 containing the c.312T>G variant. +P or -P indicates the cells were grown in the presence (+) or absence (-) of puromycin (to inhibit potential nonsense-mediated decay). NTC = no template control. The red arrowhead indicates the smaller product in the samples containing the variant. Sanger sequencing of the RT-PCR product shows that this smaller product corresponds to loss of the 22 nucleotides following the variant in the cDNA (C).

Figure 5:. Novel GLDN variants identified in a family with recurrent fetal akinesia.

(A) Pedigree showing segregation of bi-allelic VUS in GLDN. (B) Sanger sequencing showed that the heterozygous c.59T>C on gDNA (in A) appeared homozygous in muscle cDNA, suggesting loss of expression from the allele containing the essential-splice site change. (C) Detection of hGLDN and mutant hGLDN fused to an extracellular myc-tag in HEK cells in culture (red) and post-fixation and permeabilization (green). Nuclei are stained with Hoechst.

Figure 6:. A family with recurrent arthrogryposis and central involvement due to a homozygous missense variant in SMPD4.

(A) Pedigree, (B) T1 midline sagittal image (Individual II:2, neonatal MRI brain scan) showing absence of the genu and rostrum, thinning and elongation of the callosal body. Microcephaly, hypoplasia of the inferior cerebellar vermis and prominent venous sinuses are also evident. (C) T2 axial image (Individual II:2) showing simplified gyration, compensatory ventriculomegaly and absent myelination. (D) Alignment showing evolutionary conservation of the p.Pro192 residue.