Recurrent arginine substitutions in the ACTG2 gene are the primary driver of disease burden and severity in visceral myopathy

Abstract

Visceral myopathy with abnormal intestinal and bladder peristalsis includes a clinical spectrum with megacystis-microcolon intestinal hypoperistalsis syndrome and chronic intestinal pseudo-obstruction. The vast majority of cases are caused by dominant variants in ACTG2; however, the overall genetic architecture of visceral myopathy has not been well-characterized. We ascertained 53 families, with visceral myopathy based on megacystis, functional bladder/gastrointestinal obstruction, or microcolon. A combination of targeted ACTG2 sequencing and exome sequencing was used. We report a molecular diagnostic rate of 64% (34/53), of which 97% (33/34) is attributed to ACTG2. Strikingly, missense mutations in five conserved arginine residues involving CpG dinucleotides accounted for 49% (26/53) of disease in the cohort. As a group, the ACTG2-negative cases had a more favorable clinical outcome and more restricted disease. Within the ACTG2-positive group, poor outcomes (characterized by total parenteral nutrition dependence, death, or transplantation) were invariably due to one of the arginine missense alleles. Analysis of specific residues suggests a severity spectrum of p.Arg178>p.Arg257>p.Arg40 along with other less-frequently reported sites p.Arg63 and p.Arg211. These results provide genotype-phenotype correlation for ACTG2-related disease and demonstrate the importance of arginine missense changes in visceral myopathy.

Keywords: ACTG2; dysmotility; megacystis-microcolon intestinal hypoperistalsis; smooth muscle; visceral myopathy.

Figure 1 –

Molecular characterization of the cohort. (a) Apparent patterns of inheritance in the Baylor College of Medicine (BCM) Visceral Myopathy Cohort. Blue represents sporadic cases in the family. Brown represents families with multiple affected members over at least two generations suggesting a dominant inheritance pattern. Orange represents families with affected siblings born to asymptomatic parents suggesting a recessive pattern of inheritance. Red represents unknown pattern of inheritance. (b) Overall molecular diagnosis rates in the BCM Visceral Myopathy Cohort. Blue represents cases with a molecular diagnosis. Orange represents cases without a molecular diagnosis. (c) Number of cases diagnosed by different molecular strategies (exome versus targeted testing). (d) Inheritance patterns observed in the cohort. Red represents de novo ACTG2 cases. Purple represents ACTG2 cases with unknown pattern of inheritance. Blue represents MYLK (autosomal recessive). Orange represents cases lacking a molecular diagnosis. (e) Variant types in the BCM cohort. Red represents missense variants in ACTG2 affecting Arginine residues. Purple represents all other ACTG2 variants. Blue represents variants in MYLK. Orange represents undiagnosed cases.

Figure 2 –

Clinical features in the ACTG2-positive versus negative cases. (a,b) Genitourinary features (megacystis [a] and bladder decompression/catheterization [b]). Red represents cases found to harbor ACTG2 pathogenic variants. Orange represents cases without pathogenic variants in ACTG2. (c,d) Gastrointestinal features (microcolon [c] and abdominal surgery in the first weeks of life [d]). Brown represents cases with inherited ACTG2 variants or variants with unknown inheritance pattern. Red represents cases found to harbor de novo ACTG2 pathogenic variants. Orange represents cases without pathogenic variants in ACTG2.

Figure 3 –

Burden of disease attributed to ACTG2. (a) Proportion of disease complications (including megacystis, fetal bladder procedure, bilious emesis, abdominal surgery in the first weeks of life, microcolon, abnormal gastrointestinal motility study and need for bladder catheterization) that is attributed to all ACTG2 cases, ACTG2 de novo cases and ACTG2 negative cases. (b) Proportion of cases with arginine variants (blue), all other molecularly solved cases (light blue) and cases lacking a molecular diagnosis (orange) in the Baylor College of Medicine cohort. (c) Overall outcomes in the cohort. Poor outcome is defined as death in early childhood, dependence on total parenteral nutrition, or cases undergoing visceral transplant. Favorable outcome is defined as lacking these features. Brown represents proportion attributed to ACTG2 inherited cases or of unknown inheritance. Red represents ACTG2 de novo cases. Orange represents ACTG2 negative cases. (d) Disease severity in the ACTG2-positive versus ACTG2-negative. Red represents poor outcomes defined as above. Blue represents more favorable outcomes.

Figure 4 –

ACTG2 variants. (a) ACTG2 variants in the Baylor College of Medicine cohort. Bars represent number of probands with a variant at each position shown (e.g., P39 indicates proline at position 39 or p.Pro39). The amino acid change is indicated on the graph. (b) Arginine residues shown on the ACTG2 exon structure. Red labels indicate sites encoded by a “CGX” codon (CpG dinucleotides). Blue labels indicate all other Arginine residues. Red stars indicate a recurrently-mutated site. Positions of the arginine residues are labeled (e.g., R40H/C indicates p.Arg40His and p.Arg40Cys). (c) Arginine missense variants in public human databases (GnomAD and Clinvar). Black filled double circles in gnomAD indicate presence of the allele at Minor allele frequency (MAF) > 0 in the database. Black dots represent pathogenic or likely pathogenic variants in ClinVar. Red boxes represent positions encoded by CpG dinucleotides. Blue boxes represent all other arginine variants. Red stars indicate recurrent pathogenic variants in our cohort, all present in ClinVar and absent from GnomAD. Black stars indicate Arginine missense alleles that are present in both ClinVar as pathogenic or likely pathogenic variants in ClinVar which are also present at MAF > 0 in GnomAD.

Figure 5 –

Genotype-phenotype correlations for ACTG2 Arginine missense variants. Positions of the arginine residues are labeled (e.g., R40 indicates arginine at position 40 or p.Arg40). (a,b) Outcomes of arginine versus non-arginine missense variants in the Baylor College of Medicine (BCM) cohort (a) and meta-analysis incorporating cases from the BCM cohort and cases from the literature (b). Red bars represent poor outcome defined as death in early childhood, total parenteral nutrition dependence and patient undergoing visceral transplant for intestinal dysmotility. Blue bars represent cases lacking these features. (c) Outcomes of specific Arginine missense variants. Red bars represent poor outcome defined as above. Blue bars represent more favorable outcomes lacking these features.