Matera et al., “Variants of the ACTG2 gene correlate with degree of severity and presence of
megacystis in chronic intestinal pseudo-obstruction”
ONLINE SUPPLEMENTARY INFORMATION
Study subjects and clinical evaluation
To date, our cohort of CIPO patients includes 30 sporadic patients and three families. Among
familial cases, we observed evidence of autosomal dominant inheritance in one family (F1),
affected by severe constipation and recurrent episodes of intestinal obstruction with a
histopathological diagnosis of Intestinal neuronal dysplasia type B (INDB)1, and autosomal
recessive inheritance in two families, one with evidence of INDB (F2) and another one
diagnosed with familial visceral myopathy (FVM) (F3). In particular, F1 is a 2-generation
family with mother and three children, all affected. They had an histological evaluation which
showed a localized recto-colonic INDB phenotype. Family F2 presents with 2 affected
children showing an histological evaluation of INDB phenotype. In family F3, three out of
four children showed a progressive intestinal pseudo-obstruction. All the patients had an
histological evaluation of visceral myopathy with intact myenteric and submucosal plexuses.
The parents of these latter two families (F2 and F3) were healthy and non consanguineous.
Sporadic patients had either neonatal/infantile (27 probands) or adult (3 probands) onset and
were diagnosed with pseudo-obstruction (19 probands), pseudo-obstruction with megacystis
(5 probands), MMIHS (4 probands), or congenital short bowel syndrome (CSBS) (2
probands). At histopathology most patients showed the presence of giant ganglia, an INDB
hallmark1. Moreover, two patients showed a histological diagnosis of myopathy, with
glycogen vacuoles in one and associated aplastic desmosis in the other. Study recruitment
lasted for a period of 20 years.
Patient study, approved by the Ethical Committee of the Gaslini Institute, was conducted with
written informed consent collected either at the time of enrolment or, whenever possible, after
tracing back the least recent patients and families.
Whole Exome Sequencing (WES) and data analysis
Whole Exome Sequencing (WES) was applied to a first set of CIPO patients diagnosed with
INDB, including eight sporadic cases and one proband from each of two families (F1 and F2).
DNA samples were sequenced at the Beijing Genomics Institute (BGI), and successive data
analyses, shared between the Children’s Hospital of Philadelphia (CHOP) and Gaslini
Institute, were performed.
Raw data were mapped and aligned to the GRCh37 (hg19) human genome reference
assembly using the Burrows-Wheeler Aligner (BWA)2. TheSAM (Sequence Alignment Map)
files thus obtained were converted to the BAM (binary version of SAM) format and sorted by
SAMtools3. Duplicates were marked and removed by Picard tools
(https://broadinstitute.github.io/picard/) and the coverage of the samples calculated. Local
realignment around known indels and base quality score recalibration were performed by the
2.5 version of the Genome Analysis ToolKit (GATK)4 using different options including
Realigner Target Creator, Indel Realigner and Base Recalibrator on the sorted BAM files.
Variant calling was performed by the Unified Genotyper tool of GATK. The annotation of all
the variants was performed by using SnpEffect5. Softwares for functional prediction (eg. Sift
and Polyphen) and conservation degree (eg. GERP) were interrogated. Global Minor Allele
Frequencies (GMAF) were also considered in order to distinguish between rare (<1%),
uncommon (1-5%) and common (>5%) variants, as already suggested6.
Sanger sequencing
In order to confirm variants found at the ACTG2 locus, DNA amplification followed by
Sanger sequencing was carried out. Primers for the coding exons of ACTG2 were also
designed to screen 22 additional sporadic CIPO patients and one affected member of an
additional family (Table S1).
Immunoistochemistry
Tissue specimens, available from one sporadic proband, included formalin-fixed paraffinembedded blocks. Slices of 3-4 μm were stained by hematoxylin and eosin (H&E), Gomori's
trichrome stain, Gomori’s method for reticular fibers, the picrosirius red (4 μm) and periodic
acid-Schiff (PAS) techniques (without and with α-amylase digestion).
Immunohistochemical (IHC) staining was performed with the following antibodies: calretinin
(mouse monoclonal antibody, clone 5A5, Ready To Use, Leica), microtubule-associated
protein-2 (MAP-2) (mouse monoclonal antibody, clone AD-20, RTU, Thermo Scientific), S100 protein (mouse monoclonal antibody, clone S1-61-69, dilution 1:20, Leica) for ganglion
cells and nerves; desmin (mouse monoclonal antibody, clone DDE-R-11, dilution 1:100,
Leica), muscle specific actin (mouse monoclonal antibody, clone HHF35, dilution 1:200,
Leica), smooth muscle actin (SMA) (mouse monoclonal antibody, clone αsm-1, dilution 1:50,
Leica) for the muscle cells. All the IHC staining were performed using the Leica BOND-III
automated immunohistochemical stainer according to the manufacturer’s guidelines.
Bibliography
1. Knowles CH, De Giorgio R, Kapur RP et al. The London Classification of gastrointestinal
neuromuscular pathology: report on behalf of the Gastro 2009 International Working
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2. Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform.
Bioinformatics 2009;25:1754-60.
3. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G,
Durbin R; 1000 Genome Project Data Processing Subgroup. The Sequence alignment/map
(SAM) format and SAMtools. Bioinformatics 2009;25:2078-9.
4. McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Garimella K,
Altshuler D, Gabriel S, Daly M, DePristo MA. The Genome Analysis Toolkit: a
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5. Cingolani P, Platts A, Wang le L, Coon M, Nguyen T, Wang L, Land SJ, Lu X, Ruden
DM. A program for annotating and predicting the effects of single nucleotide
polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118;
iso-2; iso-3. Fly (Austin) 2012;6:80-92.
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Nutter N, Pierce EA, Blanton SH, Weinstock GM, Wilson RK, et al. Exome-Based
Mapping and Variant Prioritization for Inherited Mendelian Disorders. Am J Hum Genet
2014;94:373–84.
Table S1 Primers for molecular analysis of the coding exons of the ACTG2 gene
Name
ACTG2_F_ex2
ACTG2_R_ex2
ACTG2_F_ex3
ACTG2_R_ex3
ACTG2_F_ex4
ACTG2_R_ex4
ACTG2_F_ex5
ACTG2_R_ex5
ACTG2_F_ex6
ACTG2_R_ex6
ACTG2_F_ex7
ACTG2_R_ex7
ACTG2_F_ex8
ACTG2_R_ex8
ACTG2_F_ex9
ACTG2_R_ex9
ACTG2_F_ex4alta
ACTG2_R_ex4alta
Sequence: (5' to 3')
CCCAAAGCCAAGAAACTGTC
AAAGGGTCCTGCTCTATCAGTT
AGGCCACCACACAAGAAATC
AGGTTGGAGAAATGGTCGTG
GGCCCTGTTGAGAAGGAGTA
GGCAGGAACTATGCCAGTTT
GCTATGTCTGTGGTCCATGC
CACATGGGCATTCTCACCTA
GATGATTCTTGTGATGGGTGAA
GAGGGACTGCTTCCATGACT
GGAGGTTTTCATGGAGATCAA
GCCATGACTCCTGGTGTTTC
TGACAAGGAAGGGGGTATGA
GGATAAAATTGGCTCCAGTCC
CCATGGAAGGAATAAGGGAAG
CAGCACTATTGCATGGATTAAAA
TAGGCCACTGTGCTCTGTCA
TGAGCACTGGCCTGAGTAAA
Amplicon size (bp)
480
498
483
400
485
365
483
500
366
Primers were designated on the ACTG2 RefSeqGene (NG_034140.1), based on transcript
NM_001615.3, unless otherwise indicated
a
Transcript Uc010fey.3