Multiplex Ligation-dependent Probe Amplification Test Research Articles

Carrier Screening and Diagnosis for Spinal Muscular Atrophy Using Droplet Digital PCR Versus MLPA: Analytical Validation and Early Test Outcome.

Background: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular life-threatening disorder. Owing to high carrier frequency, population-wide SMA screening to quantify the copy number of SMN gene is recommended by American College of Medical Genetics and Genomics. An accurate, reliable, short runaround time and cost-effective method may be helpful in mass population screening for SMA. Methods: Multiplex ligation-dependent probe amplification (MLPA) is a gold standard to estimate the copy number variation (CNV) for SMN1 and SMN2 genes. In this study, we validated droplet digital polymerase chain reaction (ddPCR) for the determination of CNV for both SMN1 and SMN2 exon 7 for a diagnostic purpose. In total, 66 clinical samples were tested using ddPCR, and results were compared with the MLPA as a reference test. Results: For all samples, CNV for SMN1 and SMN2 exon 7 was consentaneous between ddPCR and MLPA test results (κ = 1.000, p < 0.0001). In addition, ddPCR also showed a significant acceptable degree of test repeatability, coefficient of variation < 4%. Conclusion: ddPCR is expected to be utilitarian for CNV detection for carrier screening and diagnosis of SMA. ddPCR test results for CNV detection for SMN1/SMN2 exon 7 are concordant with the gold standard. ddPCR is a more cost-effective and time-saving diagnostic test for SMA than MLPA. Furthermore, it can be used for population-wide carrier screening for SMA.

Indication for molecular testing by multiplex ligation-dependent probe amplification in parkinsonism.

The monogenic forms of Parkinson's disease represent less than 10% of familial cases and a still lower frequency of sporadic cases. However, guidelines to orient genetic testing are lacking. We aim to establish the interest of multiplex ligation-dependent probe amplification as a primary screening test and to propose clinical criteria to guide genetic diagnostic tests for patients with suspected Mendelian Parkinson's disease. We recruited 567 patients with parkinsonism from 547 unrelated families and performed two multiplex ligation-dependent probe amplifications for each. We confirmed all pathogenic G2019S variants in the LRRK2 gene by Sanger sequencing and screened the PRKN gene for a second mutation in cases of one heterozygous structural variant in the PRKN gene. The performance of multiplex ligation-dependent probe amplifications was 51/567 (9%) for the entire cohort and included 27 (4.8%) LRRK2 G2019S mutations, 19 (3.4%) PRKN mutations, and 5 (0.9%) SNCA locus duplications. The variables significantly associated with a positive test in the total cohort were North African ancestry (p < 0.0001), female sex (p = 0.004), and younger age at onset (p < 0.0008). Retrospective analysis allowed us to refine our indication criteria: (i) North African ancestry, (ii) an age at onset < 40, or (iii) a familial history of parkinsonism with at least one affected first-degree relative. Our study highlights the interest of MLPA testing for other parkinsonisms cases with a family history, especially for patients with dementia with Lewy bodies or a multiple system atrophy-like phenotype.

Correlation between large FBN1 deletions and severe cardiovascular phenotype in Marfan syndrome: Analysis of two novel cases and analytical review of the literature.

Marfan syndrome (MFS) is a clinically heterogeneous hereditary connective tissue disorder. Severe cardiovascular manifestations (i.e., aortic aneurysm and dissection) are the most life-threatening complications. Most of the cases are caused by mutations, a minor group of which are copy number variations (CNV), in the FBN1 gene. Multiplex ligation-dependent probe amplification test was performed to detect CNVs in 41 MFS patients not carrying disease-causing mutations in FBN1 gene. Moreover, the association was analyzed between the localization of CNVs, the affected regulatory elements and the cardiovascular phenotypes among all cases known from the literature. A large two-exon deletion (exon 46 and 47) was identified in two related patients, which was associated with a mild form of cardiovascular phenotype. Severe cardiovascular symptoms were found significantly more frequent in patients with FBN1 large deletion compared to our patients with intragenic small scale FBN1 mutation. Bioinformatic data analyses of regulatory elements located within the FBN1 gene revealed an association between the deletion of STAT3 transcription factor-binding site and cardiovascular symptoms in five out of 25 patients. Our study demonstrated that large CNVs are often associated with severe cardiovascular manifestations in MFS and the localization of these CNVs affect the phenotype severity.

Identifying Genetic Etiology in Patients with Intellectual Disability: An Experience in Public Health Services in Northeastern Brazil.

Intellectual disability (ID) is considered a common neuropsychiatric disorder that affects up to 3% of the population. The etiologic origin of ID may be genetic, environmental, and multifactorial. Chromosomopathies are relatively common among the genetic causes of ID, especially in the most severe cases and those associated with dysmorphic features. Currently, the application of new molecular cytogenetics technologies has increasingly allowed the identification of microdeletions, microduplications, and unbalanced translocations as causes of ID. The objective of this study was to investigate the etiology of ID in patients admitted to a public hospital in Northeastern Brazil. In total, 119 patients with ID who had normal karyotypes and fragile X exams participated in this study. The patients were initially physically examined for microdeletion syndromes and then tested using fluorescence in situ hybridization (FISH), multiplex ligation-dependent probe amplification (MLPA), methylation-sensitive polymerase chain reaction (MS-PCR), and chromosome microarray analysis (CMA), according to clinical suspicion. Patients with no diagnoses after FISH, MLPA, and/or MS-PCR evaluations were subsequently tested by CMA. The rate of etiologic diagnoses of ID in the current study was 28%. FISH diagnosed 25 out of 79 tested (31%), MLPA diagnosed 26 out of 79 tested (32%), MS-PCR diagnosed 7 out of 20 tested (35%), and the single nucleotide polymorphism array diagnosed 6 out of 27 tested (22%). Although the CMA is the most complete and recommended tool for the diagnosis of microdeletions, microduplications, and unbalance translocations in patients with ID, FISH, MLPA, and MS-PCR testing can be used as the first tests for specific syndromes, as long as the patients are first physically screened clinically, especially in the public health networks system in Brazil, where resources are scarce.

Severity Scoring Cutoff for MLPA and Its Diagnostic Yield in 332 North Indian Children with Developmental Delay.

Chromosomal aberrations/rearrangements are the most common cause of intellectual disability (ID), developmental delay (DD), and congenital malformations. Traditionally, karyotyping has been the investigation of choice in such cases, with the advantage of being cheap and easily accessible, but with the caveat of the inability to detect copy number variations of sizes less than 5 Mb. Chromosomal microarray can solve this problem, but again the problems of expense and poor availability are major challenges in developing countries. The purpose of this study is to find the utility of multiplex ligation-dependent probe amplification (MLPA) as a middle ground, in a resource-limited setting. We also attempted to establish an optimum cutoff for the de Vries score, to enable physicians to decide between these tests on a case-to-case basis, using only clinical data. A total of 332 children with DD/ID with or without facial dysmorphism and congenital malformations were studied by MLPA probe sets P245. Assessment of clinical variables concerning birth history, facial dysmorphism, congenital malformations, and family history was done. We also scored the de Vries scoring for all the patients to find a suitable cutoff for MLPA screening. In our study, the overall detection rate of MLPA was 13.5% (45/332). The majority of patients were DiGeorge's syndrome with probe deletion in 22q11.21 in 3.3% (11/332) followed by 15q11.2 del in 3.6% (12/332, split between Angelman's and Prader-Willi's syndromes). Also, 3.0% (10/332) of patients were positive for Williams-Beuren's syndrome 7q11.23, 1.8% (6/332) for Wolf--Hirschhorn's syndrome 4p16.3, 1.2% (4/332) for 1p36 deletion, and 1% for each trichorhinophalangeal syndrome type I 8q23.3 duplication syndrome and cri du chat syndrome. The optimum cutoff of de Vries score for MLPA testing in children with ID and/or dysmorphism came out to be 2.5 (rounded off to 3) with a sensitivity of 82.2% and specificity of 66.7%. This is the largest study from India for the detection of chromosomal aberrations using MLPA common microdeletion kit P245. Our study suggests that de Vries score with a cutoff of 3 or more can be used to offer MLPA as the first tier test for patients with unexplained ID, with or without facial dysmorphism and congenital malformations.

A pilot study of newborn screening for Duchenne muscular dystrophy in Guangzhou

BackgroundTo estimate the overall situation of Duchenne muscular dystrophy (DMD) screening in newborns in Guangzhou, China. MethodA total of 62553 newborns including 44268 males and 18285 females were screened for DMD by measuring muscle specific creatine kinase isoform (CK-MM) concentrations using the GSP® Neonatal CK-MM kit based on time-resolved immunofluorescence. We recalled positive cases and recollected dried blood spots (DBS) for retest of CK-MM. The newborns with retest positive result were recalled again for serum creatine kinase (CK) and multiplex ligation-dependent probe amplification (MLPA) test. Whole exon sequencing was performed when MLPA test was negative. ResultsFour males were diagnosed with DMD. The incidence of males was 1/11067. No DMD patient was found in female. There were significant differences of CK-MM concentration between male and female newborns. Among gestational age (GA), birth weight (BW) and age at sampling, linear regression analysis showed that CK-MM concentration was much more closely correlated with GA and age at sampling. ConclusionsCK-MM concentration is affected by gender, GA, BW and age at sampling. The efficiency of DMD screening might be improved by adjusting a multitier cut-off value according to GA and age at sampling. DMD newborn screening should be male priority.

Germline mutations in ATM, CHEK2, and other known/potential breast cancer susceptibility genes among BRCA-negative Uruguayan patients with breast cancer.

10596 Background: Breast cancer (BC) is the most frequent and the leading cause of death from cancer in Uruguayan women. In a previous study, we found BRCA1/2 germline mutations in less than 25% of the Uruguayan breast/ovarian cancer families studied. Due the impact and relevance of genetic testing in BC prevention and with the commitment to assure a wide accessibility, we developed genetic testing facilities at the University Hospital with the aim of analyzing and characterizing germline mutations in non BRCA known susceptibility and likely susceptibility genes. Methods: We studied 104 families who met the National Comprehensive Cancer Network (NCCN) criteria of hereditary BC. Analysis by next generation sequencing of BRCA1/2 genes revealed that 23 out of 104 cases studied (22%) carried pathogenic variants. Germline DNA from 42 cases BRCA negative was sequenced and analyzed for nine additional susceptibility genes (ATM, BARD1, CDH1, CHEK2, NBN, PALB2, PTEN, STK11 y TP53) included in a genetic panel. Also, in 24 cases negative for this panel we analyzed large deletions in BRCA1/2 by multiplex ligation-dependent probe amplification (MLPA) testing. In addition, to identify novel non-canonical causative genes, we extended our analysis to a complete exome sequencing of cases tested negative in genetic panels and MLPA analysis. Results: Among the 42 cases BRCA negative the sequencing of the extended genetic panel revealed 12 (28.8%) variants, 8 of uncertain significance (VUS) and 4 pathogenic, 2 novel (one in the ATM gene: NM_000051: c.614T &gt; G and other in p53: NM_000546: c.245insT) and 2 previously reported (one in PALB2: NM_024675: c.2186insA and one in PALB2/1: NM_024675c.G7T genes). Regarding the VUS, it is worth highlighting the one found in exon 12 of CHEK2 (NM_007194: c.1333T &gt; C) which was unusually frequent (16.7%) in our cohort when compared with public databases. Analysis of large deletions in BRCA1/2 in 24 cases without mutations in the genetic panel were negative for MLPA testing. Otherwise, complete exome sequencing of germline DNA from 22 cases negative for BRCA1/2 and for the genetic panel, revealed 18 variants, 16 that generate stop codons and 2 that generate loss of the termination codon (stoploss), suggesting a high potential for pathogenicity. Some striking examples are represented by the PSG2, CEP 135 y CEP 162, ZNF17 and ZNF260 genes. Conclusions: Of the 42 BRCA-negative cases, 4 (9.5%) carried a PV in a cancer susceptibility gene. The negative results obtained with MLPA in cases negative for BRCA and the extended genetic panel suggest that large deletions of BRCA are not a relevant cause of cancer susceptibility in this cohort. Otherwise, whole exome sequencing of 22 cases negatives for the previous genetic test showed pathogenic or probably PV in genes with no current evidence of association with breast and ovarian cancer.

Non-Classical Congenital Adrenal Hyperplasia-Causing Alleles in Adolescent Girls with PCOS and in Risk Group for PCOS Development.

Background: Polycystic ovary syndrome (PCOS) is the most common endocrinopathy in women. Depending on the diagnostic criteria applied, it occurs in up to 16.6% of the general female population. Congenital adrenal hyperplasia includes a group of autosomal recessive disorders, the most common of which is non-classical congenital adrenal hyperplasia (NCAH) caused by mutations in the CYP21A2 gene. PCOS and NCAH have similar clinical manifestations (hyperandrogenemia, i.e., hirsutism, acne, alopecia, and increased androgen levels in the blood) and potential impact on long-term health (infertility, increased risk of type 2 diabetes, and cardiovascular disease. Consequently, it is thought that NCAH mutations in the heterozygous state may play a role in PCOS development and phenotypic expression. Objective: To determine the prevalence of the most common pathogenic alleles of the CYP21A2 gene in adolescents with PCOS and adolescents at risk of PCOS development, and to compare the results with healthy adolescents matched for gynecological age. Methods: A cross-sectional study was conducted with 55 PCOS patients, 23 risk patients (with hyperandrogenism but a normal menstrual cycle), and 49 healthy adolescents. Genetic variations in the CYP21A2 gene were analyzed using a standard Multiplex Ligation-dependent Probe Amplification test (SALSA MLPA Probemix P050-C1 CAH; MRC Holland). Results: No significant differences were found among the three groups regarding the frequency of carriers of NCAH variations in the heterozygous state. It was found that the I172N carrier in the PCOS group had a significantly higher Global Acne Grading Scale score than PCOS patients without this variation (p = 0.038). Within the control group of healthy adolescents, compound heterozygous carriers (IVS2-12A > G and -113G > A) had a significantly higher body mass index than non-carriers (p = 0.036). Conclusion: We found no differences in the incidence of NCAH-causing variations in the heterozygous state in adolescent PCOS patients, risk adolescents (with hirsutism but normal menstruation), and healthy adolescents. Future studies of larger cohorts and rarer pathogenic CYP21A2 gene variations are required.

Germline pathogenic variant spectrum in 25 cancer susceptibility genes in Turkish breast and colorectal cancer patients and elderly controls.

Inherited pathogenic variants account for 5% to 10% of all breast cancer (BC) and colorectal cancer (CRC) cases. Here, we sought to profile the pathogenic variants in 25 cancer susceptibility genes in Turkish population. Germline pathogenic variants were screened in 732 BC patients, 189 CRC patients and 490 cancer-free elderly controls, using next-generation sequencing-based multigene panel testing and multiplex ligation-dependent probe amplification testing. Pathogenic variants were detected in 17.2% of high-risk BC patients and 26.4% of high-risk CRC patients. More than 95% of these variants were clinically actionable. BRCA1/2 and mismatch repair genes (MLH1, MSH2 and MSH6) accounted for two-thirds of all pathogenic variants detected in high-risk BC and CRC patients, respectively. Pathogenic variants in PALB2, CHEK2, ATM and TP53 were also prevalent in high-risk BC patients (4.5%). BRCA1 exons 17-18 deletion and CHEK2 c.592+3A>T were the most common variants predisposing to BC, and they are likely to be founder variants. Three frequent MUTYH pathogenic variants (c.884C>T, c.1437_1439delGGA and c.1187G>A) were responsible for all MUTYH biallelic cases (4.4% of high-risk CRC patients). The total pathogenic variant frequency was very low in controls (2.4%) and in low-risk BC (3.9%) and CRC (6.1%) patients. Our study depicts the pathogenic variant spectrum and prevalence in Turkish BC and CRC patients, guiding clinicians and health authorities for genetic testing applications and variant classification in Turkish population.

Clinical and Genetic Analyses of 38 Chinese Patients with Peutz-Jeghers Syndrome.

Background Peutz-Jeghers syndrome (PJS) is a rare autosomal dominant inherited disease caused by a germline mutation in the STK11 gene. It is characterized by mucocutaneous pigmentation, gastrointestinal hamartomatous polyps, and cancer predisposition. Aims We aimed to summarize the main clinical and genetic features of Chinese PJS patients and assessed the genotype-phenotype correlations. Methods Thirty-eight patients clinically diagnosed with Peutz-Jeghers syndrome were included in this study from 2016 to 2019. Combined direct sequencing and multiplex ligation-dependent probe amplification tests were used to detect germline heterogeneous STK11 mutations. RNA sequencing was performed in polyps of PJS patients and control groups to evaluate the difference in expression of STK11. The genotype-phenotype correlations were calculated by Kaplan-Meier analyses. Results All 26 probands and 12 affected relatives had germline heterogeneous STK11 mutations among which 8 variants were novel. Individuals with missense mutations had their first surgery and other symptoms significantly later than individuals with null mutations. Conclusion This study expanded the spectrum of STK11 gene mutations and further elucidated individuals with null mutations of STK11 typically had an earlier onset of PJS symptoms and needed earlier management.

Genotypic characteristics of Chinese patients with BHD syndrome and functional analysis of FLCN variants

BackgroundBirt-Hogg-Dubé syndrome (BHDS) is an autosomal dominant disease featured by lung cysts, spontaneous pneumothorax, fibrofolliculomas and renal tumors. The causative gene for BHDS is the folliculin (FLCN) gene and more than 200 mutations have been reported in FLCN, mostly truncating mutations. The aim of this study is to better characterize the clinical features and mutation spectrum of Chinese BHDS patients and to systematically evaluate the effects of non-truncating mutations on mRNA splicing pattern.MethodsWe enrolled 47 patients from 39 unrelated families with symptoms highly suggestive of BHDS after informed consent and detailed clinical data were collected. Exon sequencing followed by multiplex ligation-dependent probe amplification testing were applied for mutation screening. The effects of non-truncating mutations, including 15 missense mutations and 6 in-frame deletions, on mRNA splicing were investigated by minigene assays.ResultsA total of 24 FLCN germline variants were found in 39 patients from 31 distinct families. Out of these patients, 100% (36/36) presented with lung cysts and 58.3% (21/36) had experienced spontaneous pneumothorax. Seventeen mutation carriers had skin lesions (47.2%, 17/36) and 9 (30%, 9/30) had kidney lesions including 8 with renal cysts and 1 with renal hamartoma. Among all detected variants 14 (58.3%, 14/24) were novel, including 11 variants classified to be pathogenic and 3 variants of uncertain significance. None of 21 non-truncating mutations changed the mRNA splicing pattern of minigenes.ConclusionsWe found different clinical features of Chinese BHDS patients compared with Caucasians, with more lung cysts and pneumothorax but fewer skin lesions and malignant renal cancer. Chinese patients with BHDS also have a different mutation spectrum from other races. Non-truncating mutations in FLCN did not disrupt mRNA splicing pattern, in turn supporting the hypothesis that these mutations impair folliculin function by disrupting the stability of the FLCN gene product.

Hereditary Neuropathy with Liability to Pressure Palsy: A Case Diagnosed with a Quick Multiplex Ligation-dependent Probe Amplification Test

Hereditary Neuropathy with Liability to Pressure Palsy: A Case Diagnosed with a Quick Multiplex Ligation-dependent Probe Amplification Test

Diagnostic value of multiplex ligation dependent probe amplification combined with Sanger sequencing in 21-hydroxylase deficiency

Objective: To study the procedure of CYP21A2 gene mutation detection in 21-hydroxylase deficiency (21-OHD) patients. Methods: The detail clinical and biochemical data of 51 patients with 21-OHD [18 males and 33 females, with an average age of (16.4±9.9) years] were collected between December 2016 and December 2017 at Department of Endocrinology, Peking Union Medical College Hospital. Multiplex ligation dependent probe amplification (MLPA) and Sanger sequencing of the CYP21A2 gene were used to clarify the cause of 21-OHD. The genotype-phenotype correlation was also analyzed. Results: The incidences of large deletion, 8 bp deletion, I2G, I172N and F306+T were 19.6% (20/102), 1.0% (1/102), 30.4% (31/102), 25.5% (26/102) and 1.0%(1/102), respectively, and the detection rate of gene mutation in 51 21-OHD patients was 77.5% (79/102) by MLPA test. Except large and 8 bp deletion, all above mutations contained in MLPA and other 8 mutations, including P31L, Q319X, R361L, R357W, V282L, R484Q, G425S and R342W were detected, and the detection rate was 79.4% (81/102) by Sanger sequencing of CYP21A2. MLPA combined with direct sequencing identified mutations in all patients. Genotype correlated well with clinical phenotype in 21-OHD patients. Conclusions: When MLPA or CYP21A2 gene sequencing were used alone to diagnose the cause of 21-OHD, gene mutations in all patients could not be detected. The combination of the two methods can complement each other and fully clarify the underlying causes of 21-OHD.

Identification of gross deletions in FBN1 gene by MLPA

BackgroundMarfan syndrome (MFS) is an autosomal dominant connective tissue disorder caused by mutations in the FBN1 gene. Approximately 90% of classic MFS patients have a FBN1 mutation that can be identified by single-gene sequencing or gene-panel sequencing targeting FBN1. However, a small proportion of MFS patients carry a large genomic deletion in FBN1, which cannot be detected by routine sequencing. Here, we performed an MLPA (multiplex ligation-dependent probe amplification) test to detect large deletions and/or duplications in FBN1 and TGFBR2 in 115 unrelated Chinese patients with suspected MFS or early-onset aneurysm/dissection.ResultsFive novel large deletions encompassing a single exon or multiple exons in the FBN1 gene were characterized in five unrelated patients, of which four were proven by Sanger sequencing, and the breakpoints were identified. Three of them met the revised Ghent criteria when genetic results were not available, and the other two patients were highly suspected and diagnosed with MFS until the FBN1 deletions were identified.ConclusionsOur finding expands the mutation spectrum of large FBN1 deletions and emphasizes the importance of screening for large FBN1 deletions in clinical genetic testing, especially for those with classic Marfan phenotype.

Neurofibromatosis type I: mutation spectrum of NF1 in spanish patients.

Neurofibromatosis type I (NF1) is one of the most common genetic disorders in humans. NF1, a tumor predisposition syndrome, is caused by heterozygous pathogenic variants in the NF1 gene. Molecular genetic testing of NF1 is complex, especially because of the presence of a high number of partial pseudogenes, some of them with a high percentage of sequence identity. In this study, we have analyzed the largest cohort of NF1 Spanish patients (150 unrelated individuals suspected of having NF1 and 53 relatives, making a total of 203 individuals). Mutation analysis of the entire coding region was performed in all unrelated index patients. Additionally, the Multiplex Ligation-dependent Probe Amplification (MLPA) test of the NF1 gene and SPRED1 gene analysis (sequencing and MLPA test) was performed in some of the negative patients for NF1 point mutations. When fulfilling the National Institutes of Health (NIH) criterion for the clinical diagnosis of NF1, the detection rate was 79%. Among the 80 genetically confirmed NF1 probands, we detected 69 different pathogenic variants. Two mutations (3%) were gross deletions of the whole gene, the remaining 78 mutations (97%) were small changes spread among all NF1 exons. Among these 69 different mutations detected, 42 mutations were described elsewhere, and 27 mutations were novel mutations. When segregation was studied, 67% of mutations resulted de novo variants. No genetic mosaicism was detected on patients' parents.

Clinical spectrum and genetic landscape for hereditary spastic paraplegias in China

BackgroundHereditary spastic paraplegias (HSP) is a heterogeneous group of rare neurodegenerative disorders affecting the corticospinal tracts. To date, more than 78 HSP loci have been mapped to cause HSP. However, both the clinical and mutational spectrum of Chinese patients with HSP remained unclear. In this study, we aim to perform a comprehensive analysis of clinical phenotypes and genetic distributions in a large cohort of Chinese HSP patients, and to elucidate the primary pathogenesis in this population.MethodsWe firstly performed next-generation sequencing targeting 149 genes correlated with HSP in 99 index cases of our cohort. Multiplex ligation-dependent probe amplification testing was further carried out among those patients without known disease-causing gene mutations. We simultaneously performed a retrospective study on the reported patients exhibiting HSP in other Chinese cohorts. All clinical and molecular characterization from above two groups of Chinese HSP patients were analyzed and summarized. Eventually, we further validated the cellular changes in fibroblasts of two major spastic paraplegia (SPG) patients (SPG4 and SPG11) in vitro.ResultsMost patients of ADHSP (94%) are pure forms, whereas most patients of ARHSP (78%) tend to be complicated forms. In ADHSP, we found that SPG4 (79%) was the most prevalent, followed by SPG3A (11%), SPG6 (4%) and SPG33 (2%). Subtle mutations were the common genetic cause for SPG4 patients and most of them located in AAA cassette domain of spastin protein. In ARHSP, the most common subtype was SPG11 (53%), followed by SPG5 (32%), SPG35 (6%) and SPG46 (3%). Moreover, haplotype analysis showed a unique haplotype was shared in 14 families carrying c.334C > T (p.R112*) mutation in CYP7B1 gene, suggesting the founder effect. Functionally, we observed significantly different patterns of mitochondrial dynamics and network, decreased mitochondrial membrane potential (Δψm), increased reactive oxygen species and reduced ATP content in SPG4 fibroblasts. Moreover, we also found the enlargement of LAMP1-positive organelles and abnormal accumulation of autolysosomes in SPG11 fibroblasts.ConclusionsOur study present a comprehensive clinical spectrum and genetic landscape for HSP in China. We have also provided additional evidences for mitochondrial and autolysosomal-mediated pathways in the pathogenesis of HSP.

Abstract 3081: Methylation changes and somatic mutations as early events in Lynch syndrome-associated colorectal cancer

Abstract Lynch syndrome (LS) is a hereditary cancer predisposition syndrome caused by germline mutations in the DNA mismatch repair (MMR) genes resulting in an increased risk of colorectal cancer (CRC) and other malignancies. Those germline defects with other genetic and epigenetic changes accelerate multistep tumorigenesis. However, early events leading to polyp formation and the timing and order of the molecular “hits” remain unknown. Studies on sporadic CRC have indicated that promoter hypermethylation leading to gene silencing can act as an alternative mechanism to mutations at early stages of tumorigenesis, but its importance in hereditary CRC is obscure. In this study we aimed to define 1) methylation changes that occur at different stages of LS-associated colorectal tumor progression, and 2) somatic mutations that occur in adenomas comparing the status of low- and high-grade dysplasia and MMR deficiency (MMR-D) and proficiency (MMR-P). Colorectal biopsies including normal mucosae, adenomas and carcinomas were prospectively collected from 104 LS patients during colonoscopy surveillance, supplemented with retrospective tumor specimens from 56 patients. Promoter methylation was analyzed using the methylation-specific multiplex ligation-dependent probe amplification test (MS-MLPA) including selected tumor suppressor genes (TSGs) associated with early colon oncogenesis. Hypermethylation tendency was evaluated based on the frequency of CpG island methylator phenotype (CIMP) according to the methylation status of eight established CIMP marker genes. LINE-1 methylation was studied as a surrogate marker for global hypomethylation. Immunohistochemistry was used to detect MMR protein expression in neoplastic lesions. As an ongoing study mutation status of adenomas are studied using the Ion AmpliSeqTM Colon and Lung Cancer Panel targeting mutational hot spots in 22 genes. Results indicate that the expression of the MMR protein corresponding to the gene mutated in the germline decreases along with dysplasia but occurs as a relatively late event in the tumor progression, suggesting the presence of other somatic events that drive neoplastic transformation. Taken together, methylation of TSGs and frequency of CIMP increased and LINE-1 methylation decreased in adenomas and carcinomas along with dysplasia. However, a significant increase of methylation in some genes was detected already in adenomas with low-grade dysplasia compared to normal mucosae. In addition, a proportion of low-grade adenomas could already be classified CIMP positive. When comparing MMR-P and MMR-D adenomas, LINE-1 methylation decreased along the loss of MMR protein expression, and interestingly, higher methylation of SFRP1 was observed in MMR-P adenomas compared to MMR-D cases and normal mucosae. These findings emphasize the importance and early appearance of epigenetic changes in LS-associated tumorigenesis. Citation Format: Satu Mäki-Nevala, Satu Valo, Ari Ristimäki, Laura Renkonen-Sinisalo, Anna Lepistö, Jukka-Pekka Mecklin, Päivi Peltomäki. Methylation changes and somatic mutations as early events in Lynch syndrome-associated colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3081.

Detection of BRCA1 gross rearrangements by droplet digital PCR.

Large genomic rearrangements (LGRs) constitute a significant share of pathogenic BRCA1 mutations. Multiplex ligation-dependent probe amplification (MLPA) is a leading method for LGR detection; however, it is entirely based on the use of commercial kits, includes relatively time-consuming hybridization step, and is not convenient for large-scale screening of recurrent LGRs. We developed and validated the droplet digital PCR (ddPCR) assay, which covers the entire coding region of BRCA1 gene and is capable to precisely quantitate the copy number for each exon. 141 breast cancer (BC) patients, who demonstrated evident clinical features of hereditary BC but turned out to be negative for founder BRCA1/2 mutations, were subjected to the LGR analysis. Four patients with LGR were identified, with three cases of exon 8 deletion and one women carrying the deletion of exons 5-7. Excellent concordance with MLPA test was observed. Exon 8 copy number was tested in additional 720 BC and 184 ovarian cancer (OC) high-risk patients, and another four cases with the deletion were revealed; MLPA re-analysis demonstrated that exon 8 loss was a part of a larger genetic alteration in two cases, while the remaining two patients had isolated defect of exon 8. Long-range PCR and next generation sequencing of DNA samples carrying exon 8 deletion revealed two types of recurrent LGRs. Droplet digital PCR is a reliable tool for the detection of large genomic rearrangements.

The SLC22A5 genetic analysis in Chinese patients with systemic primary carnitine deficiency

Objective To investigate the clinical and biochemical metabolic features of 12 patients with systemic primary carnitine deficiency(CDSP)and to identify the SLC22A5 gene mutation types of the disease. Method The clinical and biochemical data were collected by retrospective analysis. DNA direct sequencing and multiplex ligation dependent probe amplification(MLPA)were applied for SLC22A5 gene analysis. Result Among 12 patients with CDSP, 3 cases had evident infection factors, 6 cases with convulsions, 5 cases manifested liver hypertrophy, 8 cases with hyperammonemia, and 9 cases showed myocardial damage. All CDSP patients were detected biallelic pathogenic mutation in SLC22A5 gene by direct sequencing. The gene types include IVS2+ 1G>T, c. 3G>T(p.Met1Ile), c. 760C>T(p.Arg254X), c. 1400C>G(p.Ser467Cys), c. 844dupc(p.Arg282fs), c. 338G>A(p.Cys113Tyr), c. 51C>G(p.Phe17Leu), c. 659A>T(p.Glu220Val), and c. 1365dupC(p.Thr456fs). c. 659A>T(p.Glu220Val)and c. 1365dupC(p.Thr456fs)are novel mutations. One female patient was maternal CDSP, her child had abnormal newborn screening. The allele frequency of c. 760C>T(p.Arg254X)and c. 1400C>G(p.Ser467Cys)were 37.5%(9/24)and 29.2%(7/24)respectively. The MLPA test results of all patients were negative. Conclusion The clinical manifestations are complex and various in patients with CDSP. Point and small InDel(insertions/deletions)mutation constitute the major alteration in SLC22A5 gene. c. 1400C>G(p.Ser467Cys)might be another prevalence mutation type in Chinese CDSP patient. (Chin J Endocrinol Metab, 2017, 33: 208-214) Key words: Systemic primary carnitine deficiency; SLC22A5; Free carnitine; Acylcarnitine; Multiplex ligation dependent probe amplification

Recurrent large genomic rearrangements in BRCA1 and BRCA2 in an Irish case series

Mutations in BRCA1 and BRCA2 confer a highly increased risk of cancers, mainly of the breast and ovary. Most variants are point mutations or small insertions/deletions detectable by Sanger sequencing. Large genomic rearrangements, including deletions/duplications of multiple exons, are not routinely detectable by Sanger sequencing, but can be reliably identified by Multiplex Ligation-dependent Probe Amplification (MLPA), and account for 5-17% mutations in different populations. Comprehensive mutation testing using these two methods has been facilitated via our centre since 2005. The aim of this study was to investigate the incidence of and phenotype associated with large genomic rearrangements in BRCA1 and BRCA2 in an Irish cohort. An observational cohort study was undertaken. Patients with large genomic rearrangements in BRCA1/BRCA2 were identified from a prospectively maintained database of MLPA test results. Phenotypic and genotypic data were retrieved by chart review. Large genomic rearrangements in BRCA1 were identified in 49 families; and in BRCA2 in 7 families, representing ~11% of mutations in BRCA1/BRCA2 in Ireland. The most common large genomic rearrangement in BRCA1 was deletion of exons 1-23 (11 families, 7 from Co. Galway). Other common mutations included deletions of exon 3 (8 families) and exons 1-2 (6 families). Deletion of exons 19-20 in BRCA2 represented the familial mutation in five families, all from East Ireland (Wexford/Wicklow/Dublin). It is evident that a significant proportion of highly penetrant pathogenic variants in BRCA1 and BRCA2 will be missed if testing is limited to PCR-based Sanger sequencing alone. Screening for large genomic rearrangements in BRCA1 and BRCA2 in the routine diagnostic workflow is critical to avoid false negative results.