Long Li

Cumulative Risk Impact of RET, SEMA3, and NRG1 Polymorphisms Associated with Hirschsprung Disease in Han Chinese.

Objectives: Hirschsprung disease (HSCR) is a congenital aganglionosis of myenteric and submucosal plexuses affecting a variable length of the intestine. The incidence of HSCR is approximately 1 of 5000 live births; however, the risk shows remarkable individual variation caused by single nucleotide polymorphisms (SNPs) at the RET, SEMA3, and NRG1 loci. The present study investigated the effects of these variants on the disease development and phenotype in a Chinese population. Methods: In total, 6 SNPs were genotyped in a cohort consisting of 115 patients with HSCR and 117 unaffected controls using a TaqMan genotyping assay. Histological identification of the affected-segment length (short, long, or total colonic aganglionosis) was performed for all of the samples before DNA extraction. Results: Significant genetic risk was imparted by rs2435357 and rs2506030 at RET and by rs12707682 at SEMA3. In addition, the average cumulative risk score in the patients with HSCR was significantly higher than that in the controls. Through the assessment of risk alleles by effect size, individuals were classified into 3 weighted risk score groups: low (⩽3), medium (4), and high (≥5). Individuals in the high group were significantly more susceptible to HSCR than those in the low group with an odds ratio of 7.7 (95% confidence interval 3.7–16.3). Conclusions: Cumulative genetic risk varied >35-fold between newborns with zero and >5 accumulated susceptibility alleles. The SNPs rs2435357, rs2506030, and rs12707682 may be useful for stratifying the Chinese population into distinct risk groups.

The mid-term outcomes of TRM–PIAS, proctocolectomy and ileoanal anastomosis for total colonic aganglionosis

AimsThe present study aimed to evaluate the mid-term outcomes of total colonic aganglionosis (TCA) after transanal rectal mucosectomy and partial internal anal sphincterectomy (TRM–PIAS), proctocolectomy and ileoanal anastomosis.Patients and methodsFrom 2012 to 2014, 12 patients (7 boys; 58.3xa0%) diagnosed with TCA and treated with the TRM–PIAS, proctocolectomy and ileoanal anastomosis. Seven TCA patients who underwent laparotomy-assisted endorectal pull-through (LEPT) between 2010 and 2012 were used as control group. Demographic features and complication of the two groups were evaluated. The functional outcomes were assessed by using a score system.ResultsThe procedure was successfully performed in all patients. The incidence of postoperative HAEC in the TRM–PIAS group was significantly lower (25.0 vs 85.7xa0%; pxa0<xa00.05) than control group within the second postoperative year. The number of bowel movement after 3, 12 and 24xa0months postoperatively, was 8.5xa0±xa03.5, 5.3xa0±xa02.9 and 3.1xa0±xa01.4 (pxa0<xa00.05) per day, respectively, in the TRM–PIAS group. The soiling was noted in 50.0xa0% (nxa0=xa06) of the patients in the 6th postoperative month, and 25.0xa0% (nxa0=xa03) in the 24th postoperative month in the TRM–PIAS group. There was no significant difference in overall functional outcome between two groups, but the TRM–PIAS group was better in terms of bowel movement and soiling.ConclusionTRM–PIAS, proctocolectomy and ileoanal anastomosis might be an effective treatment for TCA. More prospective studies evaluating the TRM–PIAS technique over longer period and with greater sample size are needed to confirm the findings in this study.

RET somatic mutations are underrecognized in Hirschsprung disease

PurposeWe aimed to determine the frequency of RET mosaicism in Hirschsprung disease (HSCR), test whether it has been underestimated, and to assess its contribution to HSCR risk.MethodsTargeted exome sequencing (nu2009=u200983) and RET single-gene screening (nu2009=u200969) were performed. Amplicon-based deep sequencing was applied on multiple tissue samples. TA cloning and sequencing were conducted for validation.ResultsWe identified eight de novo mutations in 152 patients (5.2%), of which six were pathogenic mosaic mutations. Two of these patients were somatic mosaics, with mutations detected in blood, colon, and saliva (mutant allele frequency: 35–44%). In addition, germ-line mosaicism was identified in four clinically unaffected subjects, each with an affected child, in multiple tissues (mutant allele frequency: 1–28%).ConclusionSomatic mutations of the RET gene are underrecognized in HSCR. Molecular investigation of the parents of patients with seemingly sporadic mutations is essential to determine recurrence risk in these families.

Annular pancreas in Trichorhinophalangeal syndrome type II with 8q23.3-q24.12 interstitial deletion

BackgroundTrichorhinophalangeal syndrome type II (TRPS II, OMIM # 150230) is a rare autosomal dominant genetic disorder characterized by craniofacial and skeletal abnormalities. Loss of functional copies of the TRPS1 gene at 8q23.3 and the EXT1 gene at 8q24.11 are considered to be responsible for the syndrome.Case PresentationHerewith, we report an 8-year-old girl with sparse scalp hair, bulbous nose, thin upper lip, broad eyebrows, phalangeal abnormalities of both hands/toes, multiple exostoses, mild intellectual impairment and severe malnutrition. In addition, the patient also had annular pancreas, a rare co-existing feature in patients with TRPS II.ConclusionsA contiguous 5.47xa0Mb deletion involving 8q23.3-q24.12 was detected by array comparative genomic hybridization (aCGH), leading to haploinsufficiency of 10 protein coding genes, 1 long non-coding RNA and 1 microRNA. Quantitative PCR (qPCR) examination confirmed half-reduced DNA copy of the patient and normal expression of both parents, indicating a de novo origin of the deletion and complete penetrance of the mutation.

Sporadic Hirschsprung Disease: Mutational Spectrum and Novel Candidate Genes Revealed by Next-generation Sequencing

Hirschsprung disease (HSCR) is a common cause of functional colonic obstruction in children. The currently available genetic testing is often inadequate as it mainly focuses on RET and several other genes, accounting for only 15–20% of cases. To identify novel, potentially pathogenic variants, we isolated a panel of genes from a whole-exome sequencing study and from the published mouse aganglionosis phenotypes, enteric nervous system development, and a literature review. The coding exons of 172 genes were analyzed in 83 sporadic patients using next-generation sequencing. Rare stop-gain, splice-site variants, frameshift and in-frame insertions/deletions and non-synonymous variants (conserved and predicted to be deleterious) were prioritized as the most promising variants to have an effect on HSCR and subjected to burden analysis. GeneMANIA interaction database was used to identify protein–protein interaction-based networks. In addition, 6 genes (PTPN13, PHKB, AGL, ZFHX3, LAMA1, and AP3B2) were prioritized for follow-up studies: both their time-space expression patterns in mouse and human colon showed that they are good candidates for predicting pathogenicity. The results of this study broaden the mutational spectrum of HSCR candidate genes, and they provide an insight into the relative contributions of individual genes to this highly heterogeneous disorder.

Response to Brosens et al

To the Editor:We appreciate this opportunity to respond to the letter “Do RET Somatic Mutations Play a Role in Hirschsprung Disease?,” by Brosens et al.,1 regarding our article2. First, we thank Brosens and colleagues for pointing out that “routine genetic testing on DNA derived from blood or saliva would not find these ENCC-specific mutations, nor would it easily detect low mosaic variants” because this is exactly why we say RET somatic mutations are underrecognized in Hirschsprung disease. This is also why we suggest that “deep sequencing (with enough sensitivity) of parental blood for pathogenic DNMs [de novo mutations] seen in children would be highly recommended, and should enable meaningful stratification of families into a substantial majority with a o1% recurrence risk and a small minority with a recurrence risk that could be at least an order of magnitude higher.” Second, we agree with the authors that “HSCR is a complex inherited disorder” and the “missing heritability seen in HSCR is a common feature of many complex disorders, and explaining it remains challenging.” We would like to emphasize that we did not discuss missing heritability in our paper, even though amplicon-based deep sequencing (ADS) indeed revealed high-frequency (75%) RET mosaicism among our cases with deleterious variants. While explaining the property of these variants, we seriously considered the usage of our terms and added “All of the six mosaic mutations we identified showed strong evidence of pathogenicity. Four are predicted to be null alleles, one has been reported previously, and one inserts an amino acid at a highly conserved site, is absent from the unaffected sibling, and has never been reported previously by the public National Heart, Lung, and Blood Institute or Exome Aggregation Consortium exome sequencing projects.” We believe that this description is objective and completely in line with American College of Medical Genetics and Genomics guidelines. It should be noted that among the eight cases with deleterious RET variants, only two (families 7 and 8) were demonstrated to be true germ-line DNM carriers. Of the remaining six, four (families 3–6) of the parents have some mosaicism, including in the germ line because the mutant allele was transmitted to the child. This simply says that there is RET mosaicism and the deleterious allele may not always be recognized when present. Besides, it’s not uncommon to use the term “somatic mosaicism” when apparently healthy parents can potentially have multiple affected children.3 With respect to the patients in families 1 and 2, we think the variants represent a postzygotic instead of germline origin for the following reasons:

Experience of Mowat–Wilson syndrome prenatal diagnosis for a Chinese family

Mowat–Wilson syndrome (MWS) is a complex developmental disorder. We report the first prenatal diagnosis provided for a family in mainland China after identifying the causal mutation for the proband. Special focus on MWS‐related organs during prenatal ultrasound scan is described which is extremely important for genetic counseling of parents.