Homozygous deletion in MYL9 expands the molecular basis of megacystis-microcolon-intestinal hypoperistalsis syndrome

Abstract

Megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS) is a severe disease characterized by functional obstruction in the urinary and gastrointestinal tract. The molecular basis of this condition started to be defined recently, and the genes related to the syndrome (ACTG2-heterozygous variant in sporadic cases; and MYH11 (myosin heavy chain 11), LMOD1 (leiomodin 1) and MYLK (myosin light chain (MLC) kinase)-autosomal recessive inheritance), encode proteins involved in the smooth muscle contraction, supporting a myopathic basis for the disease. In the present article, we described a family with two affected siblings with MMIHS born to consanguineous parents and the molecular investigation performed to define the genetic etiology. Previous whole exome sequencing of the affected child and parents did not identify a candidate gene for the disease in this family, but now we present a reanalysis of the data that led to the identification of a homozygous deletion encompassing the last exon of MYL9 (myosin regulatory light chain 9) in the affected individual. MYL9 gene encodes a regulatory myosin MLC and the phosphorylation of this protein is a crucial step in the contraction process of smooth muscle cell. Despite the absence of human or animal phenotype related to MYL9, a cause-effect relationship between MYL9 and the MMIHS seems biologically plausible. The present study reveals a strong candidate gene for autosomal recessive forms of MMIHS, expanding the molecular basis of this disease and reinforces the myopathic basis of this condition.

Fig. 1

Pedigree and molecular investigation of the homozygous deletion in MYL9 gene. a Pedigree shows two affected siblings with MMIHS born from consanguineous parents (F = 16), and identifies the individuals molecularly tested. b Integrative Genomics Viewer (IGV) view of WES trio (patient and her parents) showing no reads in exon 4 of the MYL9 gene in the patient and a reduced number of reads in both parents suggesting a homozygous and heterozygous deletions, respectively. *There are no reads in exon 1 of the MYL9 gene, because it was not included in the capture. c Electrophoresis showing the PCR amplicon of exon 4 (407 bp) in parents and control subject. No amplification was observed in the patient. d Electrophoresis showing PCR amplicon (954 bp) using primers 2F–5R that were designed to amplify the boundaries of deletion breakpoints in the patient and her parents. No amplification of the wild-type fragment (7918 bp) was observed in the control individual because the protocol used for the PCR does not amplify fragments > 5 kb. e Sanger sequencing of amplicon shown in d identifies a deletion of 6964 bp (chr20:g.36548744_36555707del). The chromatogram illustrates the breakpoints of the deletion: the sequence agrees with the reference until the genomic coordinate chr20:g.36548743 (flanking region—green box I). After that, the reference sequence (flanking region—black box) is replaced by amplicon sequence which align from coordinate chr20:g.36555708 to chr20:g.36556186 (flanking region—green box II)

Fig. 2

Strategy used to define the breakpoints of the deletion encompassing the last exon of MYL9 gene (exon 4) 5′-region: primers named 1F and 1R (blue arrows) were designed to anneal in a genomic region upstream from the last probe detected by CytoScan^® HD array. The subsequent primer pairs (2F and 2R—red arrows; 3F and 3R—yellow arrows) were designed toward the deleted region (2F is complementary and reverse to 1R, 3F is complementary and reverse to 2R); 3′-region: primers named as 6F and 6R (gray arrows) were designed to anneal in a genomic region downstream from the first probe detected by CytoScan^® HD array. The primers pairs named 4F and 4R (orange arrows) and 5F and 5R (green arrows) were designed toward the deleted region (4R is complementary and reverse to 5F, 5R is complementary and reverse to 6F). PCR amplification occurred for all primer pairs in the control individual. No PCR amplification was observed using 2F–2R, 3F–3R, 4F–4R and 5F–5R primers in the patient sample; however, PCR was successful for primers 1F–1R and 6F–6R (not shown). Amplicon containing the deleted region was obtained using primers 2F and 5R. This amplicon was submitted to Sanger sequencing. F forward primer, R reverse primer

Fig. 3

Contraction of smooth muscle cell: cellular pathway and the role of MYL9 and of other genes related to MMIHS. a Diagram illustrating some steps in the cellular pathway related to smooth muscle contraction. The phosphorylation of regulatory myosin light chain (rMLC) encoded by MYL9 is crucial to promote contraction and occurs at the end of this pathway (thick arrow). The increase in intracellular calcium concentration [Ca²⁺]i is an essential step to initiate the process, which is triggered by several factors (autonomic nervous system, hormones, local chemical, or mechanical stimulus). The [Ca²⁺]i increases due to two mechanisms: the influx of calcium ions (Ca²⁺) from extracellular to cytosol mediated by different channels (Ca²⁺ voltage-dependent and nonselective cation channels) and by the release of this ion from sarcoplasmatic reticulum (SR) through a G-protein- (G) mediated cascade. The binding of agonists to the G-protein-coupled receptor increases the phospholipase C (PLC) activity via G-protein leading to the production of inositol 1,4,5-triphophate (IP3) and diacylglycerol (DAG) from membrane phospholipids—phosphatidylinositol 4,5-biphosphate (PIP2). Protein G also activates the Rho pathway. IP3 binds to a specific receptor (IP3R) on SR causing the release of Ca²⁺ to the cytosol. The activation of the ryanodine receptor (RyR) on SR also contributes to the release of Ca²⁺ to the cytosol and both, IPR3 and RyR, are activated by increased [Ca²⁺]i (not shown). The intracellular Ca²⁺ (four molecules) binds to calmodulin (CaM) and this complex activates the myosin light chain kinase (MLCK). Activated MLCK phosphorylates the rMLC of myosin class II (also composed by myosin heavy chain—MHC and essential myosin light chain—eMLC), allowing actin–myosin binding and generating the force necessary to contract the cell. Dephosphorylation of rMLC occurs by the action of the myosin light chain phosphatase (MLCP), inducing the relaxation. MLCP is inactivated via phosphorylation mediated by kinases (PKC, RhoK, and ZIPK—activated by DAG and Rho G pathway) and activated by the cyclic nucleotide-dependent pathway. The increase in [Ca²⁺]i also stimulates the contraction by the pathway related to caldesmon, an actin-binding protein (not shown). b The presumed effect in smooth muscle contraction due to deletion in MYL9: the disruption of rMLC caused by deletion impairs the phosphorylation mediated by MLCK, leading to reduced or absent contraction. c The genes and the respective contractile filaments and inheritance patterns related to MMIHS—ACTG2 filamentous actin (F-actin), gamma-2 isoform, MYH11 myosin heavy chain (MHC) 11, MYLK myosin light chain kinase (MLCK), MYL9 regulatory myosin light chain (rMLC), LMOD1 leiomodin 1, an actin-binding protein. *eMLC encoded by MYL6 gene, not related to MMIHS until now, AD autosomal dominant inheritance, AR autosomal recessive inheritance. The information regarding smooth muscle contraction was based on different references [, –30]