Exome sequencing for diagnosis of congenital hemolytic anemia.

BACKGROUND: Congenital hemolytic anemia constitutes a heterogeneous group of rare genetic disorders of red blood cells. Diagnosis is based on clinical data, family history and phenotypic testing, genetic analyses being usually performed as a late step. In this study, we explored 40 patients with congenital hemolytic anemia by whole exome sequencing: 20 patients with hereditary spherocytosis and 20 patients with unexplained hemolysis. RESULTS: A probable genetic cause of disease was identified in 82.5% of the patients (33/40): 100% of those with suspected hereditary spherocytosis (20/20) and 65% of those with unexplained hemolysis (13/20). We found that several patients carried genetic variations in more than one gene (3/20 in the hereditary spherocytosis group, 6/13 fully elucidated patients in the unexplained hemolysis group), giving a more accurate picture of the genetic complexity of congenital hemolytic anemia. In addition, whole exome sequencing allowed us to identify genetic variants in non-congenital hemolytic anemia genes that explained part of the phenotype in 3 patients. CONCLUSION: The rapid development of next generation sequencing has rendered the genetic study of these diseases much easier and cheaper. Whole exome sequencing in congenital hemolytic anemia could provide a more precise and quicker diagnosis, improve patients' healthcare and probably has to be democratized notably for complex cases.

CFTR and/or pancreatitis susceptibility genes mutations as risk factors of pancreatitis in cystic fibrosis patients?

BACKGROUND/OBJECTIVES: Currently, factors that promote the occurrence of pancreatitis episodes in patients affected with cystic fibrosis (CF) and pancreatic sufficiency (PS) are largely unknown. METHODS: Six genes involved in pancreatitis or in ion transport into the pancreatic duct were investigated by next generation sequencing in 59 adult CF-PS patients with two identified CF mutations. Data on predisposing environmental factors were also recorded. RESULTS: 19 experienced at least one episode of acute pancreatitis (AP) (AP+) and 40 patients did not (AP-). No influence of environmental factor was evidenced. No specific CFTR genotype was found predictive of pancreatitis. Patients sharing the same CFTR genotype may or may not experience AP episodes. Frequent and rare missense variants were found in 78.9% patients in group AP+ and 67.5% in group AP- but a few of them were pathogenic. CONCLUSIONS: AP or recurrent AP (RAP) is a frequent complication in our series of adult CF-PS patients. The majority of mild CFTR mutations found in group AP+ were located in the first transmembrane region. No clear other genetic factor could be found predictive of AP/RAP. Further experiments in large homogenous cohorts of CF-PS patients, including whole genome sequencing, may identify genetic predisposing factors to pancreatitis.

Identification of a novel 5' alternative CFTR mRNA isoform in a patient with nasal polyposis and CFTR mutations.

Cystic fibrosis may be revealed by nasal polyposis (NP) starting early in life. We performed cystic fibrosis transmembrane conductance regulator (CFTR) DNA and mRNA analyses in the family of a 12-year-old boy presenting with NP and a normal sweat test. Routine DNA analysis only showed the heterozygous c.2551C>T (p.Arg851*) mutation in the child and the father. mRNA analysis showed partial exon skipping due to c.2551C>T and a significant increase in total CFTR mRNA in the patient and the mother, which was attributable to the heterozygous c. -2954G>A variant in the distant promoter region, as demonstrated by in vitro luciferase assays. The 5' rapid amplification of cDNA ends analysis showed the presence of a novel transcript, where the canonical exon 1 was replaced by an alternative exon called 1a-Long. This case report could represent the first description of a CFTR-related disorder associated with the presence of a 5' alternative, probably nonfunctional transcript, similar to those of fetal origin.

A recurrent deep-intronic splicing CF mutation emphasizes the importance of mRNA studies in clinical practice.

BACKGROUND: The identification by CFTR mRNA studies of a new deep-intronic splicing mutation, c.870-1113_1110delGAAT, in one patient of our series with mild CF symptoms and in three CF patients of an Italian study, led us to evaluate the mutation frequency and phenotype/genotype correlations. METHODS: 266 patients with CF and related disorders and having at least one undetected mutation, were tested at the gDNA level in three French reference laboratories. RESULTS: In total, the mutation was found in 13 unrelated patients (5% of those already carrying a mutation) plus 4 siblings, including one homozygote and 12 heterozygotes having a severe CF mutation. The sweat test was positive in 10/14 documented cases, the diagnosis was delayed after 20 years in 9/15 and pancreatic insufficiency was present in 5/16. CONCLUSION: c.870-1113_1110delGAAT should be considered as CF-causing with phenotype variability and overall delayed diagnosis. Its frequency highlights the potential of mRNA studies.

Comprehensive description of CFTR genotypes and ultrasound patterns in 694 cases of fetal bowel anomalies: a revised strategy.

Fetal bowel anomalies may reveal cystic fibrosis (CF) and the search for CF transmembrane conductance regulator (CFTR) gene mutations is part of the diagnostic investigations in such pregnancies, according to European recommendations. We report on our 18-year experience to document comprehensive CFTR genotypes and correlations with ultrasound patterns in a series of 694 cases of fetal bowel anomalies. CFTR gene analysis was performed in a multistep process, including search for frequent mutations in the parents and subsequent in-depth search for rare mutations, depending on the context. Ultrasound patterns were correlated with the genotypes. Cases were distinguished according to whether they had been referred directly to our laboratory or after an initial testing in another laboratory. A total of 30 CF fetuses and 8 cases compatible with CFTR-related disorders were identified. CFTR rearrangements were found in 5/30 CF fetuses. 21.2% of fetuses carrying a frequent mutation had a second rare mutation, indicative of CF. The frequency of CF among fetuses with no frequent mutation was 0.43%. Correlation with ultrasound patterns revealed a significant frequency of multiple bowel anomalies in CF fetuses. The results emphasize the need to search for rearrangements in the diagnosis strategy of fetal bowel anomalies. The diagnostic value of ultrasound patterns combining hyperechogenic bowel, loop dilatation and/or non-visualized gallbladder reveals a need to revise current strategies and to offer extensive CFTR gene testing when the triad is diagnosed, even when no frequent mutation is found in the first-step analysis.

Alternative splicing at a NAGNAG acceptor site as a novel phenotype modifier.

Approximately 30% of alleles causing genetic disorders generate premature termination codons (PTCs), which are usually associated with severe phenotypes. However, bypassing the deleterious stop codon can lead to a mild disease outcome. Splicing at NAGNAG tandem splice sites has been reported to result in insertion or deletion (indel) of three nucleotides. We identified such a mechanism as the origin of the mild to asymptomatic phenotype observed in cystic fibrosis patients homozygous for the E831X mutation (2623G>T) in the CFTR gene. Analyses performed on nasal epithelial cell mRNA detected three distinct isoforms, a considerably more complex situation than expected for a single nucleotide substitution. Structure-function studies and in silico analyses provided the first experimental evidence of an indel of a stop codon by alternative splicing at a NAGNAG acceptor site. In addition to contributing to proteome plasticity, alternative splicing at a NAGNAG tandem site can thus remove a disease-causing UAG stop codon. This molecular study reveals a naturally occurring mechanism where the effect of either modifier genes or epigenetic factors could be suspected. This finding is of importance for genetic counseling as well as for deciding appropriate therapeutic strategies.

Prognostic value of translocation t(11;18) in tumoral response of low-grade gastric lymphoma of mucosa-associated lymphoid tissue type to oral chemotherapy.

PURPOSE: To determine the impact of translocation t(11;18) on response to oral alkylating agents in gastric mucosa-associated lymphoid tissue lymphoma (GML). PATIENTS AND METHODS: Fifty-three patients with a GML were studied. Helicobacter pylori-positive patients (n = 34) received anti-H pylori treatment and H pylori-negative patients (n = 19) or patients who failed to respond to anti-H pylori treatment received oral alkylating agents. t(11;18) was detected by reverse transcription polymerase chain reaction from frozen gastric biopsies. RESULTS: t(11;18) was detected in 32% of patients. It was more prevalent in H pylori-negative as compared with H pylori-positive patients (12 of 19 v five of 34 patients; P = .0005). Among 31 H pylori-eradicated patients, t(11;18) was detected in three patients, all of whom experienced treatment failure, and it was absent in 28 patients: 21 patients (75%) were in remission and seven patients (25%) experienced treatment failure (P = .03). Among 21 patients who received an alkylating agent, t(11;18) was detected in 12 patients: five patients (42%) were in remission and seven patients (58%) experienced treatment failure. t(11;18) was absent in nine patients: eight patients (89%) were in remission and one patient (11%) experienced treatment failure by the end of treatment. Four patients in remission relapsed during follow-up (median, 7 years): they all had t(11;18). Durable remission was obtained in eight (89%) of the nine patients without t(11;18) versus one of the 12 patients (8%) with t(11;18) (P = .0003). CONCLUSION: Presence of t(11;18) in GML is predictive of resistance to oral alkylating agents, with less than 10% of durable remission at long-term follow-up.