Identification of an AluY-mediated deletion of exon 5 in the CPOX gene by MLPA analysis in patients with hereditary coproporphyria

Hereditary coproporphyria (HCP) is an autosomal dominantly inherited hepatic porphyria, caused by a mutation in the coproporphyrinogen oxidase (CPOX) gene. The genetic defect leads to a partial defect of CPOX, the sixth enzyme involved in haem biosynthesis. Affected individuals can develop acute life-threatening attacks of neurovisceral symptoms and/or more rarely cutaneous symptoms such as skin fragility and blistering. The identification of the genetic defect in HCP families is of crucial importance to detect the carrier status which allows counselling to prevent possible triggering factors, e.g. certain drugs, alcohol, or fasting. In a total of nine Swedish HCP families, routine gene sequence analysis had identified a causative mutation in only five. In the present study, using an in-house developed synthetic probe set for multiplex ligation-dependent probe amplification (MLPA) analysis, we detected a deletion of the fifth exon in the CPOX gene in the remaining four families. The deletion is 3381 bp in size and has originated by an Alu-mediated mechanism. This finding emphasizes the usefulness of MLPA analysis as a complement to gene sequencing for comprehensive genetic diagnostics in HCP patients.     

Spastin mutation screening in Chinese patients with pure hereditary spastic paraplegia

BackgroundHereditary spastic paraplegia (HSP) is a clinically and genetically heterogeneous group of neurodegenerative diseases. Mutations in the spastin (SPAST) gene are the most common cause of pure HSP. However, few data are available regarding the clinical and genetic spectrum of HSP among Chinese patients.MethodsClinical data were collected at diagnosis and follow-up of 42 Chinese patients with pure HSP. All seventeen exons of the SPAST gene were directly sequenced. Additionally, we used a multiplex ligation dependent probe amplification (MLPA) assay targeting the SPAST gene to evaluate large exon deletion or insertion mutations in patients without SPAST point mutations.ResultsThe age of disease onset of our patients was 19.6 ± 14.4 years. Six novel variations were found, including three missense mutations (p. L363P, p. D441V, and p. S595R), one insertion (c.1511dupT (p. Y505Ifs*7)), and two larger deletions (exons 5–17 and exons 10–17). Four previously reported mutations, including p. S399L, c.1215_c.1219delTATAA (p. N405Kfs*36), exon 1 deletion, and exon 16 deletion, were detected. The SPAST mutation rate was 40% (4/10) in Chinese familial patients and 33.33% (7/21) in Chinese sporadic pure HSP patients. The frequency of large deletions was high in both AD-HSP (20%, 2/10) and sporadic HSP (14.28%, 3/21).ConclusionSPAST mutations are common in Chinese patients with pure HSP. Large exon deletions are an important cause of AD-HSP and sporadic pure HSP in Chinese patients. Large fragment tests should be performed to explore large SPAST mutations in familial and sporadic HSP patients without SPAST point mutations.     

Genotype/phenotype analysis in Chinese laminin‐α2 deficient congenital muscular dystrophy patients

Laminin‐α2 deficient congenital muscular dystrophy (CMD) is an autosomal recessive disorder characterized by severe muscular dystrophy, which is typically associated with abnormal white matter. In this study, we assessed 43 CMD patients with typical white matter abnormality and laminin‐α2 deficiency (complete or partial) diagnosed by immunohistochemistry to determine the clinical and molecular genetic characteristics of laminin‐α2 deficient CMD. LAMA2 gene mutation analysis was performed by direct sequencing of genomic DNAs. Exonic deletion or duplication was identified by multiplex ligation‐dependent probe amplification (MLPA) and verified by high‐density oligonucleotide‐based CGH microarrays. Gene mutation analysis revealed 86 LAMA2 mutations (100%); 15 known and 37 novel. Among these mutations, 73.9% were nonsense, splice‐site or frameshift and 18.8% were deletions of one or more exons. Genetic characterization of affected families will be valuable in prenatal diagnosis of CMD in the Chinese population.     

Characterization of large deletions in the DHCR7 gene

Pathogenic variants in the DHCR7 gene cause Smith–Lemli–Opitz syndrome (SLOS), a defect of cholesterol biosynthesis resulting in an autosomal recessive congenital metabolic malformation disorder. In approximately 4% of patients, the second mutation remains unidentified. In this study, 12 SLOS patients diagnosed clinically and/or by elevated 7‐dehydrocholesterol (7‐DHC) have been investigated by customized multiplex ligation‐dependent probe amplification (MLPA) analysis, because only one DHCR7 sequence variant has been detected. Two unrelated patients of this cohort carry different large deletions in the DHCR7 gene. One patient showed a deletion of exons 3–6. The second patient has a deletion of exons 1 and 2 (non‐coding) and lacks the major part of the promoter. These two patients show typical clinical and biochemical phenotypes of SLOS. Second disease‐causing mutations are p.(Arg352Trp) and p.(Thr93Met), respectively. Deletion breakpoints were characterized successfully in both cases. Such large deletions are rare in the DHCR7 gene but will resolve some of the patients in whom a second mutation has not been detected.     

TP53 alterations in relapsed childhood acute lymphoblastic leukemia

TP53 alterations are frequent relapse‐acquired mutations in childhood acute lymphoblastic leukemia (ALL). The present study evaluated the clinical significance of relapsed childhood ALL in Taiwan. Diagnostic and/or relapsed bone marrow or peripheral blood was obtained from 111 children with relapsed ALL who were initially treated by using Taiwan Pediatric Oncology Group (TPOG) ALL protocols from January 1997 to May 2018. Mutations were detected by PCR and sequencing, as well as by multiplex ligation‐dependent probe amplification to detect copy number alterations. Copy number and/or sequence alterations of TP53 were detected in 29% (28 of 98) and in 46% (6 of 13) of patients with relapsed B‐cell and T‐cell ALL, respectively. This incidence was much higher than that in several similar studies conducted in Caucasian populations. Seventy percent of all TP53 alterations were gained at relapse in 67 matched samples by back‐tracking matched diagnostic samples. TP53 alterations were associated with lower 5‐year event‐free survival (EFS) and overall survival (OS) rates (P = .013 and P = .0002, respectively). Multivariate analysis confirmed the prognostic significance of TP53 alterations. Forty‐five patients received hematopoietic stem‐cell transplantations post‐relapse. Patients with TP53 alterations (14/45) had inferior 5‐year EFS and OS than patients without TP53 alterations after transplantation (P = .002 and P = .001, respectively). The significance of these TP53 alterations for patients who received transplantations was confirmed by multivariate analysis. In conclusion, TP53 alterations were enriched and useful as prognostic markers in relapsed childhood ALL.     

Frequency and Correlation of Common Genes Copy Number Alterations in Childhood Acute Lymphoblastic Leukemia with Prognosis

Objective: It was shown by genomic profiling that despite no detectable chromosomal abnormalities a proportion of children with pre-B acute lymphoblastic leukemia harbors copy number alterations (CNA) of genes playing role in B-cell development and function. The aim of the study was to determine the frequency of CNA in pediatric acute lymphoblastic leukemia and correlate these findings with clinical outcome. Methods: DNA extracted from peripheral blood or bone marrow at diagnosis/relapse of fifty newly diagnosed children with precursor B-cell acute lymphoblastic leukemia was analyzed for CNA with multiplex ligation-dependent probe amplification. Results: The analysis revealed 76 CNA in 24 patients most frequently found in PAR1 (17%), CDKN2A/B (15.7%) and PAX5 (14.4%) genes. There were significant CNA co-occurrences between PAX5, CDKN2A/B, BTG1, ETV6, PAR1 or XP22 genes, (p <0.020) and the high-risk group. There was a significant correlation between EBF1, RB1, and IKZF1 alterations and bone marrow relapse. Patients with CNA in screened genes are more likely to succumb to their disease except for those with PAR1 or XP22 genes (p <0.050). Conclusion: The multiplex ligation-dependent probe amplification could be considered as an independent diagnostic tool allowing prompt identification of patients at high risk of treatment failure and, subsequently, a more adequate treatment approach.     

The Mutation Spectrum and Two Novel Point Mutations in the APC Gene in Vietnamese Patients with Familial Adenomatous Polyposis

Background: Familial adenomatous polyposis (FAP) is a hereditary disorder primarily caused by germline mutations in the APC gene. The most common type of mutation in the APC gene is point mutation, while deletion mutation is much less frequent. The current study was conducted to investigate the mutation spectrum of the APC gene in Vietnamese FAP patients. Methods: Patients with the clinical diagnosis of FAP on colorectal endoscopy were screened for mutations in the APC gene using Sanger sequencing. Those who exhibited no point mutation subsequently underwent MLPA assay to detect deletion and duplication mutations. Besides, the relatives of patients with mutated APC genes were recruited for detecting carrier status. Results: Sixty-three patients with clinical colorectal polyposis were recruited. Mutations in the APC gene were detected in 26/63 patients (41.3%). Genetic analysis of 105 asymptomatic relatives of these 26 patients found mutations in the APC gene in 55 individuals (52.4%). Conclusion: We successfully established the APC gene mutation spectrum in Vietnamese FAP patients for the first time. Of importance, we discovered two novel point mutations in the APC gene. The high prevalence of carrier status in asymptomatic family members of patients with mutation emphasizes the crucial role of appropriate genetic screening for early diagnosis, surveillance, and preventive measurements.     

Absence of large intragenic rearrangements in the DPYD gene in a large cohort of colorectal cancer patients treated with 5-FU-based chemotherapy

AIMS

To study the relationship between the toxicity associated with a 5-FU-based therapy and the presence of (i) the large intragenic rearrangements in the DPYD gene and (ii) the IVS14+1G>A mutation.

METHODS

We used the multiplex ligation-dependent probe amplification technique (MLPA) to study genomic DNA from 234 colorectal cancer patients treated with 5-FU-based chemotherapy.

RESULTS

We did not detect any deletion/duplication in the DPYD gene. The presence of the IVS14+1G>A mutation was also excluded.

CONCLUSIONS

Neither the large genomic rearrangements in the DPYD gene nor the IVS14+1G>A mutation play a significant role in the development of serious toxicity associated with a 5-FU containing regimen.     

MLPA mutation detection in Argentine HNPCC and FAP families

Colorectal cancer (CC) is the secondary cause of death in the Western countries of which approximately 15% are considered to be hereditary. The hereditary forms are Familial Adenomatous Polyposis (FAP) and Hereditary Non Polyposis Colorectal Cancer (HNPCC) which is the commonest form. The detection of mutations in the MMR and apc related genes, allows the development of health prevention strategies. Different molecular diagnostic strategies are available for the detection of mutations in these genes, i.e. DGGE, SSCP and direct sequencing. However, deletions and duplications of one or more consecutive exons, which account for around 50% of the total alterations in MMR genes, cannot be detected by PCR based methodologies due to the non quantitative nature of these techniques. The aim of our work has been the standardization of a methodology, called Multiplex Ligation-Dependent Probe Amplification, which allows the detection of genomic deletions and duplications as primary analysis in HNPCC and FAP patients in Argentina. In this case, we inform that the application of MLPA allowed the detection of a missence mutation, without the need for direct sequencing of the complete genes involved. A PCR/RFLP strategy was afterwards designed to detect the C<T change on codon 718 of mlh1 gene in members of the family. For a developing country like Argentina, which has limited resources for genetic diagnosis, this MLPA application has avoided an unaffordable cost as the complete sequencing of all the involved genes. The application of MLPA in our country contributes to improvement in the diagnosis of hereditary CC and allows the development of preventive health interventions.     

Large <Emphasis Type="Italic">BRCA1</Emphasis> and <Emphasis Type="Italic">BRCA2</Emphasis> genomic rearrangements in Danish high risk breast-ovarian cancer families

BRCA1 and BRCA2 germ-line mutations predispose to breast and ovarian cancer. Large genomic rearrangements of BRCA1 account for 0–36% of all disease causing mutations in various populations, while large genomic rearrangements in BRCA2 are more rare. We examined 642 East Danish breast and/or ovarian cancer patients in whom a deleterious mutation in BRCA1 and BRCA2 was not detected by sequencing using the multiplex ligation-dependent probe amplification (MLPA) assay. We identified 15 patients with 7 different genomic rearrangements, including a BRCA1 exon 5–7 deletion with a novel breakpoint, a BRCA1 exon 13 duplication, a BRCA1 exon 17–19 deletion, a BRCA1 exon 3–16 deletion, and a BRCA2 exon 20 deletion with a novel breakpoint as well as two novel BRCA1 exon 17–18 and BRCA1 exon 19 deletions. The large rearrangements in BRCA1 and BRCA2 accounted for 9.2% (15/163) of all BRCA1 and BRCA2 mutations in East Denmark. Nine patients had the exon 3–16 deletion in BRCA1. By SNP analysis we find that the patients share a 5 Mb fragment of chromosome 17, including BRCA1, indicating that the exon 3–16 deletion represents a Danish founder mutation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.