Alagille syndrome (ALGS, OMIM #118450) is an autosomal dominant disorder that affects multiple organ systems including the liver, heart, eyes, vertebrae, and face. ALGS is caused by mutations in one of two genes in the Notch Signaling Pathway, JAGGED1 or NOTCH2. In this study, analysis of 21 Vietnamese ALGS individuals led to the identification of 19 different mutations (18 JAGGED1 and 1 NOTCH2), 17 of which are novel, including the third reported NOTCH2 mutation in Alagille Syndrome. The spectrum of JAGGED1 mutations in the Vietnamese patients is similar to that previously reported, including nine frameshift, three missense, two splice site, one nonsense, two whole gene, and onw partial gene deletion. The missense mutations are all likely to be disease causing, as two are loss of cysteines (C22R and C78G) and the third creates a cryptic splice site in exon 9 (G386R). No correlation between genotype and phenotype was observed. Assessment of clinical phenotype revealed that skeletal manifestations occur with a higher frequency than in previously reported Alagille cohorts. Facial features were difficult to assess and a Vietnamese pediatric gastroenterologist was only able to identify the facial phenotype in 61% of the cohort. To assess the agreement among North American dysmorphologists at detecting the presence of ALGS facial features in the Vietnamese patients, 37 clinical dysmorphologists evaluated a photographic panel of 20 Vietnamese children with and without ALGS. The dysmorphologists were unable to identify the individuals with ALGS in the majority of cases, suggesting that evaluation of facial features should not be used in the diagnosis of ALGS in this population. This is the first report of mutations and phenotypic spectrum of ALGS in a Vietnamese population.
Alagille Syndrome; JAGGED1; NOTCH2; JAGGED1 missense mutation
The utilization of genome-wide chromosomal microarray analysis (CMA) in pediatric clinical practice provides an opportunity to consider how genetic diagnostics is evolving, and to prepare for the clinical integration of genome-wide sequencing technologies. We conducted semi-structured interviews with 15 healthcare providers (7 genetic counselors, 4 medical geneticists, and 4 non-genetics providers) to investigate the impact of CMA on clinical practice, and implications for providers, patients and families. Interviews were analyzed qualitatively using content analysis. Most providers reported that genomic testing enhanced their professional experience and was beneficial to patients, primarily due to the improved diagnostic rate compared with earlier chromosomal studies. Other effects on practice included moving towards genotype-first diagnosis and broadening indications for chromosomal testing. Opinions varied concerning informed consent and disclosure of results. The duty to disclose incidental findings (IFs) was noted; however concerns were raised about potential psychosocial harms of disclosing pre-symptomatic findings. Tensions were revealed between the need for comprehensive informed consent for all families and the challenges of communicating time-consuming and potentially anxiety-provoking information regarding uncertain and incidental findings that may be relevant only in rare cases. Genetic counselors can play an important role in liaising with families, health professionals and testing laboratories, providing education and guidance to non-genetics providers, and enabling families to receive adequate pre- and post-test information and follow-up care.
Genomics; Uncertainty; Incidental findings; Clinical pediatrics; Qualitative
Alagille syndrome (ALGS) is an autosomal dominant condition, primarily caused by mutations in JAGGED1. ALGS is defined by cholestatic liver disease, cardiac disease and involvement of the face, skeleton and eyes with variable expression of these features. Renal involvement has been reported though not formally described. The objective of this study was to systematically characterize the renal involvement in ALGS.
We performed a retrospective review of 466 JAGGED1 mutation-positive ALGS patients. Charts were reviewed for serum biochemistries, renal ultrasounds or other imaging, urinalysis and clinical reports from pediatric nephrologists. The clinical data were reviewed by two pediatric hepatologists and a pediatric nephrologist.
Of 466 charts reviewed we found 187 yielded evaluable renal information. Of these, 73/187 were shown to have renal involvement, representing 39% of the study cohort. Renal dysplasia was the most common anomaly seen. Genotype analysis of the JAGGED1 mutations in the patients with and without renal involvement did not reveal an association with mutation type.
From the study we concluded that renal involvement has a prevalence of 39% in ALGS in our evaluable patients. Renal dysplasia is the most common renal anomaly. This finding correlates with the known role of the Notch pathway in glomerular development. Since renal disease of the type seen in ALGS can impair growth and impact liver transplantation, there is a clear need for a prospective study of renal involvement in ALGS and the development of guidelines for evaluation and management. These data also suggest that renal involvement be considered the sixth defining criterion for ALGS.
liver disease; renal disease; Alagille syndrome kidney
Conditions associated with sudden cardiac arrest/death (SCA/D) in youth often have a genetic etiology. While SCA/D is uncommon, a pro-active family screening approach may identify these inherited structural and electrical abnormalities prior to symptomatic events and allow appropriate surveillance and treatment. This study investigated the diagnostic utility of exome sequencing (ES) by evaluating the capture and coverage of genes related to SCA/D.
Samples from 102 individuals (13 with known molecular etiologies for SCA/D, 30 individuals without known molecular etiologies for SCA/D and 59 with other conditions) were analyzed following exome capture and sequencing at an average read depth of 100X. Reads were mapped to human genome GRCh37 using Novoalign, and post-processing and analysis was done using Picard and GATK. A total of 103 genes (2,190 exons) related to SCA/D were used as a primary filter. An additional 100 random variants within the targeted genes associated with SCA/D were also selected and evaluated for depth of sequencing and coverage. Although the primary objective was to evaluate the adequacy of depth of sequencing and coverage of targeted SCA/D genes and not for primary diagnosis, all patients who had SCA/D (known or unknown molecular etiologies) were evaluated with the project’s variant analysis pipeline to determine if the molecular etiologies could be successfully identified.
The majority of exons (97.6 %) were captured and fully covered on average at minimum of 20x sequencing depth. The proportion of unique genomic positions reported within poorly covered exons remained small (4 %). Exonic regions with less coverage reflect the need to enrich these areas to improve coverage. Despite limitations in coverage, we identified 100 % of cases with a prior known molecular etiology for SCA/D, and analysis of an additional 30 individuals with SCA/D but no known molecular etiology revealed a diagnostic answer in 5/30 (17 %). We also demonstrated 95 % of 100 randomly selected reported variants within our targeted genes would have been picked up on ES based on our coverage analysis.
ES is a helpful clinical diagnostic tool for SCA/D given its potential to successfully identify a molecular diagnosis, but clinicians should be aware of limitations of available platforms from technical and diagnostic perspectives.
Electronic supplementary material
The online version of this article (doi:10.1186/s40246-015-0038-y) contains supplementary material, which is available to authorized users.
Mosaic trisomy 17 is rare with only 28 cases reported and the clinical presentation is highly variable. The diagnosis is most commonly made by prenatal karyotype and in most cases is followed by a normal postnatal karyotype on blood lymphocytes. We present two cases of mosaic trisomy 17 diagnosed prenatally, with follow up in multiple tissues at birth. In the first case, trisomy 17 was identified in all amniocytes, and at birth standard results of chromosome analysis in peripheral blood were normal, but mosaic trisomy 17 was identified (50–75%) in skin fibroblasts by genome-wide SNP array analysis. This patient presented with minor anomalies, congenital heart disease, asymmetry, intestinal malrotation and died on day 9 of life. In the second patient amniocentesis after ultrasound finding of tetralogy of Fallot showed mosaic trisomy 17. Postnatally, results of a SNP array were normal in blood, buccal mucosa and skin. It is possible that the cardiac defect is related to trisomy 17 in key tissues during heart development, although at birth the aneuploidy could not be identified in tissues that are routinely analyzed for diagnosis. These cases add to our understanding of mosaic trisomy 17, highlighting the failure to diagnose this aneuploidy in peripheral blood.
Mosaic trisomy 17; SNP microarray analysis; tissue specific mosaicism
Mutations in the Notch pathway ligand Jagged1 (JAG1) cause Alagille syndrome (AGS), as well as cardiac defects in seemingly non-syndromic, individuals. To estimate the frequency of JAG1 mutations in cases with right-sided cardiac defects not otherwise diagnosed with AGS, we screened 94 cases with tetralogy of Fallot (TOF) and 50 with pulmonic stenosis/peripheral pulmonary stenosis (PS/PPS) or pulmonary valve atresia with intact ventricular septum (PA) for mutations. Sequence changes were identified in three TOF and three PS/PPS/PA patients,that were not present in 100 controls. We identified one frameshift and two missense mutations in the TOF cases, and one frameshift and two missense mutations in cases with PS/PPS/PA. The four missense mutations were assayed for their effect on protein localization, post-translational modification and ability to activate Notch signaling. The missense mutants displayed heterogeneous behavior in these assays, some with complete haploinsufficiency, suggesting that there are additional modifiers leading to organ specific features. We identified functionally significant mutations in 3% (2/94) of TOF patients and 4% (2/50) of PS/PPS/PA patients. Patients with right-sided cardiac defects should be carefully screened for features of AGS or a family history of cardiac defects that might suggest the presence of a JAG1 mutation.
Alagille syndrome; AGS; heart; variable expressivity; tetralogy of Fallot; pulmonary stenosis; Jagged1; JAG1; Notch signaling
Biliary atresia (BA) is a progressive, idiopathic obliteration of the extrahepatic biliary system occurring exclusively in the neonatal period. It is the most common disease leading to liver transplantation in children. The etiology of BA is unknown, although infectious, immune and genetic causes have been suggested. While the recurrence of BA in families is not common, there are more than 30 multiplex families reported and an underlying genetic susceptibility has been hypothesized. We screened a cohort of 35 BA patients for genomic alterations that might confer susceptibility to BA. DNA was genotyped on the Illumina Quad550 platform, which analyzes over 550,000 single nucleotide polymorphisms (SNPs) for genomic deletions and duplications. Areas of increased and decreased copy number were compared to those found in control populations. In order to identify regions that could serve as susceptibility factors for BA, we searched for regions that were found in BA patients, but not in controls. We identified two unrelated BA patients with overlapping heterozygous deletions of 2q37.3. Patient 1 had a 1.76 Mb (280 SNP), heterozygous deletion containing thirty genes. Patient 2 had a 5.87 Mb (1,346 SNP) heterozygous deletion containing fifty-five genes. The overlapping 1.76 Mb deletion on chromosome 2q37.3 from 240,936,900 to 242,692,820 constitutes the critical region and the genes within this region could be candidates for susceptibility to BA.
Biliary atresia; copy number variation; deletion 2q37.3
The use of array technology to define chromosome deletions and duplications is bringing us closer to establishing a genotype/phenotype map of genomic copy number alterations. We studied 21 patients and 5 relatives with deletions of the short arm of chromosome 20 using the Illumina HumanHap550 SNP array to 1) more accurately determine the deletion sizes, 2) identify and compare breakpoints, 3) establish genotype/phenotype correlations and 4) investigate the use of the HumanHap550 platform for analysis of chromosome deletions. Deletions ranged from 95kb to 14.62Mb, and all of the breakpoints were unique. Eleven patients had deletions between 95kb and 4Mb and these individuals had normal development, with no anomalies outside of those associated with Alagille syndrome. The proximal and distal boundaries of these eleven deletions constitute a 5.4MB region, and we propose that haploinsufficiency for only 1 of the 12 genes in this region causes phenotypic abnormalities. This defines the JAG1 associated critical region, in which deletions do not confer findings other than those associated with Alagille syndrome. The other 10 patients had deletions between 3.28Mb and 14.62Mb, which extended outside the critical region, and notably, all of these patients, had developmental delay. This group had other findings such as autism, scoliosis and bifid uvula. We identified 47 additional polymorphic genome-wide copy number variants (>20 SNPs), with 0–5 variants called per patient. Deletions of the short arm of chromosome 20 are associated with relatively mild and limited clinical anomalies. The use of SNP arrays provides accurate high-resolution definition of genomic abnormalities.
SNP array analysis; 20p deletion; copy number variants; Alagille syndrome; haploinsufficiency; JAG1
Pallister Killian syndrome (OMIM: # 601803) is a rare multisystem disorder typically caused by tissue limited mosaic tetrasomy of chromosome 12p (isochromosome 12p). The clinical manifestations of Pallister Killian syndrome are variable with the most common findings including craniofacial dysmorphia, hypotonia, cognitive impairment, hearing loss, skin pigmentary differences and epilepsy. Isochromosome 12p is identified primarily in skin fibroblast cultures and in chorionic villus and amniotic fluid cell samples and may be identified in blood lymphocytes during the neonatal and early childhood period. We performed genomic expression profiling correlated with interphase fluorescent in situ hybridization and single nucleotide polymorphism array quantification of degree of mosaicism in fibroblasts from 17 Caucasian probands with Pallister Killian syndrome and 9 healthy age, gender and ethnicity matched controls. We identified a characteristic profile of 354 (180 up- and 174 down-regulated) differentially expressed genes in Pallister Killian syndrome probands and supportive evidence for a Pallister Killian syndrome critical region on 12p13.31. The differentially expressed genes were enriched for developmentally important genes such as homeobox genes. Among the differentially expressed genes, we identified several genes whose misexpression may be associated with the clinical phenotype of Pallister Killian syndrome such as downregulation of ZFPM2, GATA6 and SOX9, and overexpression of IGFBP2.
This report describes an algorithm developed to predict the pathogenicity of copy number variants (CNVs) in large sample cohorts. CNVs (genomic deletions and duplications) are found in healthy individuals and in individuals with genetic diagnoses, and differentiation of these two classes of CNVs can be challenging and usually requires extensive manual curation. We have developed PECONPI, an algorithm to assess the pathogenicity of CNVs based on gene content and CNV frequency. This software was applied to a large cohort of patients with genetically heterogeneous non-syndromic hearing loss to score and rank each CNV based on its relative pathogenicity. Of 636 individuals tested, we identified the likely underlying etiology of the hearing loss in 14 (2%) of the patients (1 with a homozygous deletion, 7 with a deletion of a known hearing loss gene and a point mutation on the trans allele and 6 with a deletion larger than 1 Mb). We also identified two probands with smaller deletions encompassing genes that may be functionally related to their hearing loss. The ability of PECONPI to determine the pathogenicity of CNVs was tested on a second genetically heterogenous cohort with congenital heart defects (CHDs). It successfully identified a likely etiology in 6 of 355 individuals (2%). We believe this tool is useful for researchers with large genetically heterogeneous cohorts to help identify known pathogenic causes and novel disease genes.
Copy number variation; genetic heterogeneity
Exome sequencing is a promising method for diagnosing patients with a complex phenotype. However, variant interpretation relative to patient phenotype can be challenging in some scenarios, particularly clinical assessment of rare complex phenotypes. Each patient’s sequence reveals many possibly damaging variants that must be individually assessed to establish clear association with patient phenotype. To assist interpretation, we implemented an algorithm that ranks a given set of genes relative to patient phenotype. The algorithm orders genes by the semantic similarity computed between phenotypic descriptors associated with each gene and those describing the patient. Phenotypic descriptor terms are taken from the Human Phenotype Ontology (HPO) and semantic similarity is derived from each term’s information content.
Model validation was performed via simulation and with clinical data. We simulated 33 Mendelian diseases with 100 patients per disease. We modeled clinical conditions by adding noise and imprecision, i.e. phenotypic terms unrelated to the disease and terms less specific than the actual disease terms. We ranked the causative gene against all 2488 HPO annotated genes. The median causative gene rank was 1 for the optimal and noise cases, 12 for the imprecision case, and 60 for the imprecision with noise case. Additionally, we examined a clinical cohort of subjects with hearing impairment. The disease gene median rank was 22. However, when also considering the patient’s exome data and filtering non-exomic and common variants, the median rank improved to 3.
Semantic similarity can rank a causative gene highly within a gene list relative to patient phenotype characteristics, provided that imprecision is mitigated. The clinical case results suggest that phenotype rank combined with variant analysis provides significant improvement over the individual approaches. We expect that this combined prioritization approach may increase accuracy and decrease effort for clinical genetic diagnosis.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2105-15-248) contains supplementary material, which is available to authorized users.
Clinical; Phenotype; Exome; Genome; Informatics
Here we describe three subjects with mosaic genome-wide paternal uniparental isodisomy (GWpUPD) each of whom presented initially with overgrowth, hemihyperplasia (HH), and hyperinsulinism (HI). Due to the severity of findings and the presence of additional features, SNP array testing was performed, which demonstrated mosaic GWpUPD. Comparing these individuals to 10 other live-born subjects reported in the literature, the predominant phenotype is that of pUPD11 and notable for a very high incidence of tumor development. Our subjects developed non-metastatic tumors of the adrenal gland, kidney, and/or liver.AllthreesubjectshadpancreatichyperplasiaresultinginHI. Notably, our subjects to date display minimal features of other diseases associated with paternal UPD loci. Both children who survived the neonatal period have displayed near-normal cognitive development, likely due to a favorable tissue distribution of the mosaicism.Tounderstandthe rangeofUPDmosaicismlevels, we studied multiple tissues using SNP array analysis and detected levels of 5–95%, roughly correlating with the extent of tissue involvement.Giventherapidityoftumorgrowthandthedifficulty distinguishing malignant and benign tumors in these GWpUPD subjects, we have utilized increased frequency of ultrasound (US) and alpha-fetoprotein (AFP) screening in the first years of life. Because of a later age of onset of additional tumors, continued tumor surveillance into adolescence may need to be considered in these rare patients.
Beckwith–Wiedemann; uniparental disomy; isodisomy; mosaicism; methylation; hyperinsulinism
We report on a patient with early onset pediatric bilateral pheochromocytomas caused by mosaic chromosome 11p15 paternal uniparental isodisomy (UPD). Hemihyperplasia of the arm was diagnosed in a 4-month-old female and clinical methylation testing for 11p15 in the blood was normal, with a reported detection threshold for mosaicism of 20%. She was subsequently diagnosed at 18 months with bilateral pheochromocytomas. Single-nucleotide polymorphism (SNP) array analysis of pheochromocytoma tissue demonstrated mosaic deletions of 8p12pter, 21q21.1qter, 22q11.23qter; commonly seen in pheochromocytomas. In addition, mosaic 11p15.3pter homozygosity was noted. Molecular testing for other causes of pheochromocytomas was normal, suggesting that 11p15 homozygosity was the primary event. Subsequent SNP array analysis of skin fibroblasts from the hyperplastic side demonstrated 5% mosaic paternal UPD for 11p15. We have subsequently used SNP array analysis to identify four patients with subtle hemihyperplasia with low-level mosaic UPD that was not detected by methylation analysis. Given the increased sensitivity of SNP array analysis to detect UPD along with the increased incidence of tumorigenesis in these UPD patients, we suggest that it has high utility in the clinical work-up of hemihyperplasia. The present case also suggests that 11p15 paternal UPD may be an under-detected mechanism of sporadic pheochromocytoma in the pediatric population. Furthermore, a review of the literature suggests that patients with 11p15 paternal UPD may present after 8 years of age with pheochromocytoma and raises the possibility that ultrasound screening could be considered beyond 8 years of age in this subset of hemihyperplasia and Beckwith–Wiedemann syndrome patients.
hemihyperplasia; hemihypertrophy; Beckwith–Wiedemann; uniparental disomy; isodisomy; mosaicism; methylation; pheochromocytoma
BACKGROUND & AIMS
Biliary atresia (BA) is a progressive fibroinflammatory disorder of infants involving the extrahepatic and intrahepatic biliary tree. Its etiology is unclear but is believed to involve exposure of a genetically susceptible individual to certain environmental factors. BA occurs exclusively in the neonatal liver, so variants of genes expressed during hepatobiliary development could affect susceptibility. Genome-wide association studies previously identified a potential region of interest at 2q37. We continued these studies to narrow the region and identify BA susceptibility genes.
We searched for copy number variants that were increased among patients with BA (n = 61) compared with healthy individuals (controls; n = 5088). After identifying a candidate gene, we investigated expression patterns of orthologues in zebrafish liver and the effects of reducing expression, with morpholino antisense oligonucleotides, on biliary development, gene expression, and signal transduction.
We observed a statistically significant increase in deletions at 2q37.3 in patients with BA that resulted in deletion of one copy of GPC1, which encodes glypican 1, a heparan sulfate proteoglycan that regulates Hedgehog signaling and inflammation. Knockdown of gpc1 in zebrafish led to developmental biliary defects. Exposure of the gpc1 morphants to cyclopamine, a Hedgehog antagonist, partially rescued the gpc1-knockdown phenotype. Injection of zebrafish with recombinant Sonic Hedgehog led to biliary defects similar to those of the gpc1 morphants. Liver samples from patients with BA had reduced levels of apical GPC1 in cholangiocytes compared with samples from controls.
Based on genetic analysis of patients with BA and zebrafish, GPC1 appears to be a BA susceptibility gene. These findings also support a role for Hedgehog signaling in the pathogenesis of BA.
GWA; Susceptibility Loci; Animal Model; Bile Duct Growth and Development
Alagille syndrome (ALGS) is an autosomal dominant disorder characterized by developmental abnormalities in several organs including the liver, heart, eyes, vertebrae, kidneys, and face. The majority (90-94 %) of ALGS cases are caused by mutations in the JAG1 (JAGGED1) gene, and in a small percent of patients (∼1 %) mutations in the NOTCH2 gene have been described. Both genes are involved in the Notch signaling pathway. To date, over 440 different JAG1 gene mutations and ten NOTCH2 mutations have been identified in ALGS patients. The present study was conducted on a group of 35 Polish ALGS patients and revealed JAG1 gene mutations in 26 of them. Twenty-three different mutations were detected including 13 novel point mutations and six large deletions affecting the JAG1 gene. Review of all mutations identified to date in individuals from Poland allowed us to propose an effective diagnostic strategy based on the mutations identified in the reported patients of Polish descent. However, the distribution of mutations seen in this cohort was not substantively different than the mutation distribution in other reported populations.
Alagille syndrome; Diagnostic strategy; JAG1 gene; JAG1 point mutations; Large deletions
As genomic and exomic testing expands in both the research and clinical arenas, determining whether, how, and which incidental findings to return to the ordering clinician and patient becomes increasingly important. Although opinion is varied on what should be returned to consenting patients or research participants, most experts agree that return of medically actionable results should be considered. There is insufficient evidence to fully inform evidence-based clinical practice guidelines regarding return of results from genome-scale sequencing, and thus generation of such evidence is imperative, given the rapidity with which genome-scale diagnostic tests are being incorporated into clinical care. We present an overview of the approaches to incidental findings by members of the Clinical Sequencing Exploratory Research network, funded by the National Human Genome Research Institute, to generate discussion of these approaches by the clinical genomics community. We also report specific lists of “medically actionable” genes that have been generated by a subset of investigators in order to explore what types of findings have been included or excluded in various contexts. A discussion of the general principles regarding reporting of novel variants, challenging cases (genes for which consensus was difficult to achieve across Clinical Sequencing Exploratory Research network sites), solicitation of preferences from participants regarding return of incidental findings, and the timing and context of return of incidental findings are provided.
actionability; actionable genes; clinical sequencing; genomic medicine; incidental findings
Chromosomal microarray analysis (CMA) has improved the diagnostic rate of genomic disorders in pediatric populations, but can produce uncertain and unexpected findings. This paper explores clinicians’ perspectives and identifies challenges in effectively interpreting results and communicating with families about CMA. Responses to an online survey were obtained from 40 clinicians who had ordered CMA. Content included practice characteristics and perceptions, and queries about a hypothetical case involving uncertain and incidental findings. Data were analyzed using non-parametric statistical tests. Clinicians’ comfort levels differed significantly for explaining uncertain, abnormal, and normal CMA results, with lowest levels for uncertain results. Despite clinical guidelines recommending informed consent, many clinicians did not consider it pertinent to discuss the potential for CMA to reveal information concerning biological parentage or predisposition to late-onset disease, in a hypothetical case. Many non-genetics professionals ordering CMA did not feel equipped to interpret the results for patients, and articulated needs for education and access to genetics professionals. This exploratory study highlights key challenges in the practice of genomic medicine, and identifies needs for education, disseminated practice guidelines, and access to genetics professionals, especially when dealing with uncertain or unexpected findings.
Cytogenomics; incidental findings; uncertainty; health communication; pediatrics; genomic medicine; genetic counseling
Lymphedema-cholestasis syndrome (LCS; Aagenaes syndrome) is a rare autosomal recessive disorder, characterized by 1) neonatal intrahepatic cholestasis, often lessening and becoming intermittent with age, and 2) severe chronic lymphedema, mainly lower limb. LCS was originally described in a Norwegian kindred in which a locus, LCS1, was mapped to a 6.6cM region on chromosome 15. Mutations in CCBE1 on chromosome 18 have been reported in some cases of lymphatic dysplasia, but not in LCS.
Consanguineous parents of Mexican ancestry had a child with LCS who did not exhibit extended homozygosity in the LCS1 region. A subsequent pregnancy was electively terminated due to fetal hydrops. We performed whole-genome single nucleotide polymorphism genotyping to identify regions of homozygosity in these siblings, and sequenced promising candidate genes.
Both siblings harbored a homozygous mutation in CCBE1, c.398 T>C, predicted to result in the missense change p.L133P. Regions containing known ‘cholestasis genes’ did not demonstrate homozygosity in the LCS patient.
Mutations in CCBE1 may yield a phenotype not only of lymphatic dysplasia, but also of LCS or fetal hydrops; however, the possibility that the sibling with LCS also carries a homozygous mutation in an unidentified gene influencing cholestasis cannot be excluded.
We present a rare case of mosaicism for a structural abnormality of chromosome 12 in a patient with phenotypic features of Pallister-Killian syndrome. A six-month-old child with dysmorphic features, exotropia, hypotonia, and developmental delay was mosaic for both a normal karyotype and a cell line with 12p duplication/triplication in 25 percent of metaphase cells. Utilization of fluorescence in situ hybridization (FISH) identified three copies of probes from the end of the short arm of chromosome 12 (TEL(12p13) locus and the subtelomere (12p terminal)) on the structurally abnormal chromosome 12. Genome-wide SNP array analysis revealed that the regions of duplication and triplication were of maternal origin. The abnormal cell line in our patient was present at 25 percent at six months and 19 months of age in both metaphase and interphase cells from peripheral blood, where typically the isochromosome 12p is absent in the newborn. This may suggest that the gene(s) resulting in a growth disadvantage of abnormal cells in peripheral blood of patients with tetrasomy 12p may not have the same influence when present in only three copies.
We describe 19 unrelated individuals with submicroscopic deletions involving 10p15.3 characterized by chromosomal microarray (CMA). Interestingly, to our knowledge, only two individuals with isolated, submicroscopic 10p15.3 deletion have been reported to date; however, only limited clinical information is available for these probands and the deleted region has not been molecularly mapped. Comprehensive clinical history was obtained for 12 of the 19 individuals described in this study. Common features among these 12 individuals include: cognitive/behavioral/developmental differences (11/11), speech delay/language disorder (10/10), motor delay (10/10), craniofacial dysmorphism (9/12), hypotonia (7/11,), brain anomalies (4/6) and seizures (3/7). Parental studies were performed for nine of the 19 individuals; the 10p15.3 deletion was de novo in seven of the probands, not maternally inherited in one proband and inherited from an apparently affected mother in one proband. Molecular mapping of the 19 individuals reported in this study has identified two genes, ZMYND11 (OMIM# 608668) and DIP2C (OMIM# 611380) (UCSC Genome Browser), mapping within 10p15.3 which are most commonly deleted. Although no single gene has been identified which is deleted in all 19 individuals studied, the deleted region in all but one individual includes ZMYND11 and the deleted region in all but one other individual includes DIP2C. There is not a clearly identifiable phenotypic difference between these two individuals and the size of the deleted region does not generally predict clinical features. Little is currently known about these genes complicating a direct genotype/phenotype correlation at this time. These data however, suggest that ZMYND11 and/or DIP2C haploinsufficiency contributes to the clinical features associated with 10p15 deletions in probands described in this study.
chromosomal microarray (CMA); 10p15.3; deletion; ZMYND11; DIP2C
Mutations in Notch signaling pathway members cause developmental phenotypes that affect the liver, skeleton, heart, eye, face, kidney, and vasculature. Notch associated disorders include the autosomal dominant, multi-system, Alagille syndrome caused by mutations in both a ligand (Jagged1 (JAG1)) and receptor (NOTCH2) and autosomal recessive spondylocostal dysostosis, caused by mutations in a ligand (Delta-like-3 (DLL3)), as well as several other members of the Notch signaling pathway. Mutations in NOTCH2 have also recently been connected to Hajdu-Cheney syndrome, a dominant disorder causing focal bone destruction, osteoporosis, craniofacial morphology and renal cysts. Mutations in the NOTCH1 receptor are associated with several types of cardiac disease and mutations in NOTCH3 cause the dominant adult onset disorder CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy), a vascular disorder with onset in the 4th or 5th decades. Studies of these human disorders and their inheritance patterns and types of mutations reveal insights into the mechanisms of Notch signaling.
Alagille syndrome; Spondylocostal dysostosis; Hajdu Cheney; Cardiac disease; Notch signaling
Hearing loss is the most prevalent sensory perception deficit in humans, affecting 1/500 newborns, can be syndromic or nonsyndromic and is genetically heterogeneous. Nearly 80% of inherited nonsyndromic bilateral sensorineural hearing loss (NBSNHI) is autosomal recessive. Although many causal genes have been identified, most are minor contributors, except for GJB2, which accounts for nearly 50% of all recessive cases of severe to profound congenital NBSNHI in some populations. More than 60% of children with a NBSNHI do not have an identifiable genetic cause. To identify genetic contributors, we genotyped 659 GJB2 mutation negative pediatric probands with NBSNHI and assayed for copy number variants (CNVs). After identifying 8 mild-moderate NBSNHI probands with a Chr15q15.3 deletion encompassing the Stereocilin (STRC) gene amongst this cohort, sequencing of STRC was undertaken in these probands as well as 50 probands and 14 siblings with mild-moderate NBSNHI and 40 probands with moderately severe-profound NBSNHI who were GJB2 mutation negative. The existence of a STRC pseudogene that is 99.6% homologous to the STRC coding region has made the sequencing interpretation complicated. We identified 7/50 probands in the mild-moderate cohort to have biallelic alterations in STRC, not including the 8 previously identified deletions. We also identified 2/40 probands to have biallelic alterations in the moderately severe-profound NBSNHI cohort, notably no large deletions in combination with another variant were found in this cohort. The data suggest that STRC may be a common contributor to NBSNHI among GJB2 mutation negative probands, especially in those with mild to moderate hearing impairment.
Bilateral sensorineural hearing loss; SNHI; Chr15q15.3; Stereocilin; STRC; DFNB16; SNP genotyping array; copy number variation; CNV
The increased sensitivity of chromosomal microarray (CMA) technology as compared with traditional cytogenetic analysis allows for improved detection of genomic alterations. However, there is potential for uncertainty in the interpretation of test results in some cases. This paper explores how families understand and make meaning of CMA test results, and identifies the needs of families undergoing CMA testing.
We conducted semistructured interviews with parents of 25 pediatric outpatients with CMA test results indicating either a pathogenic alteration or a variant of unknown significance (VUS). Interviews were analyzed qualitatively.
Three domains of understanding were identified: comprehension of results, interpretations of scientific uncertainty, and personal meaning for the child and family. Incomplete comprehension of test results and scientific uncertainty were prominent themes for families receiving results in both the VUS and pathogenic categories. Receiving results from non-geneticists and by telephone, long waits to see a geneticist, and misleading Internet searches all contributed to misunderstandings.
Differentiating domains of understanding allows for the identification of uncertainties that can be reduced or managed in order to improve understanding of CMA results. Using this framework, we suggest interventions to promote clarity and address the informational needs of families undergoing CMA testing.
comprehension; genetic testing; health communication; qualitative; uncertainty
The breakpoints and mechanisms of ring chromosome formation were studied and mapped in 14 patients.
Several techniques were performed such as genome-wide array, MLPA (Multiplex Ligation-Dependent Probe Amplification) and FISH (Fluorescent in situ Hybridization).
The ring chromosomes of patients I to XIV were determined to be, respectively: r(3)(p26.1q29), r(4)(p16.3q35.2), r(10)(p15.3q26.2), r(10)(p15.3q26.13), r(13)(p13q31.1), r(13)(p13q34), r(14)(p13q32.33), r(15)(p13q26.2), r(18)(p11.32q22.2), r(18)(p11.32q21.33), r(18)(p11.21q23), r(22)(p13q13.33), r(22)(p13q13.2), and r(22)(p13q13.2). These rings were found to have been formed by different mechanisms, such as: breaks in both chromosome arms followed by end-to-end reunion (patients IV, VIII, IX, XI, XIII and XIV); a break in one chromosome arm followed by fusion with the subtelomeric region of the other (patients I and II); a break in one chromosome arm followed by fusion with the opposite telomeric region (patients III and X); fusion of two subtelomeric regions (patient VII); and telomere-telomere fusion (patient XII). Thus, the r(14) and one r(22) can be considered complete rings, since there was no loss of relevant genetic material. Two patients (V and VI) with r(13) showed duplication along with terminal deletion of 13q, one of them proved to be inverted, a mechanism known as inv-dup-del. Ring instability was detected by ring loss and secondary aberrations in all but three patients, who presented stable ring chromosomes (II, XIII and XIV).
We concluded that the clinical phenotype of patients with ring chromosomes may be related with different factors, including gene haploinsufficiency, gene duplications and ring instability. Epigenetic factors due to the circular architecture of ring chromosomes must also be considered, since even complete ring chromosomes can result in phenotypic alterations, as observed in our patients with complete r(14) and r(22).