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1.  Clinical utility of multiplex ligation-dependent probe amplification technique in identification of aetiology of unexplained mental retardation: A study in 203 Indian patients 
Background & objectives:
Developmental delay (DD)/mental retardation also described as intellectual disability (ID), is seen in 1-3 per cent of general population. Diagnosis continues to be a challenge at clinical level. With the advancement of new molecular cytogenetic techniques such as cytogenetic microarray (CMA), multiplex ligation-dependent probe amplification (MLPA) techniques, many microdeletion/microduplication syndromes with DD/ID are now delineated. MLPA technique can probe 40-50 genomic regions in a single reaction and is being used for evaluation of cases with DD/ID. In this study we evaluated the clinical utility of MLPA techniques with different probe sets to identify the aetiology of unexplained mental retardation in patients with ID/DD.
Methods:
A total of 203 randomly selected DD/ID cases with/without malformations were studied. MLPA probe sets for subtelomeric regions (P070/P036) and common microdeletions/microduplications (P245-A2) and X-chromosome (P106) were used. Positive cases with MLPA technique were confirmed using either fluorescence in situ hybridization (FISH) or follow up confirmatory MLPA probe sets.
Results:
The overall detection rate was found to be 9.3 per cent (19 out of 203). The detection rates were 6.9 and 7.4 per cent for common microdeletion/microduplication and subtelomeric probe sets, respectively. No abnormality was detected with probe set for X-linked ID. The subtelomeric abnormalities detected included deletions of 1p36.33, 4p, 5p, 9p, 9q, 13q telomeric regions and duplication of 9pter. The deletions/duplications detected in non telomeric regions include regions for Prader Willi/Angelman regions, Williams syndrome, Smith Magenis syndrome and Velocardiofacial syndrome.
Interpretation & conclusions:
Our results show that the use of P245-A2 and P070/P036-E1 probes gives good diagnostic yield. Though MLPA cannot probe the whole genome like cytogenetic microarray, due to its ease and relative low cost it is an important technique for evaluation of cases with DD/ID.
PMCID: PMC3994742  PMID: 24604040
Common microdeletion/microduplication syndromes; developmental delay; intellectual disability; India; MLPA; subtelomeric abnormalities
2.  Mutation screening of melatonin-related genes in patients with autism spectrum disorders 
BMC Medical Genomics  2010;3:10.
Background
One consistent finding in autism spectrum disorders (ASD) is a decreased level of the pineal gland hormone melatonin and it has recently been demonstrated that this decrease to a large extent is due to low activity of the acetylserotonin O-methyltransferase (ASMT), the last enzyme in the melatonin synthesis pathway. Moreover, mutations in the ASMT gene have been identified, including a splice site mutation, that were associated with low ASMT activity and melatonin secretion, suggesting that the low ASMT activity observed in autism is, at least partly, due to variation within the ASMT gene.
Methods
In the present study, we have investigated all the genes involved in the melatonin pathway by mutation screening of AA-NAT (arylalkylamine N-acetyltransferase), ASMT, MTNR1A, MTNR1B (melatonin receptor 1A and 1B) and GPR50 (G protein-coupled receptor 50), encoding both synthesis enzymes and the three main receptors of melatonin, in 109 patients with autism spectrum disorders (ASD). A cohort of 188 subjects from the general population was used as a comparison group and was genotyped for the variants identified in the patient sample.
Results
Several rare variants were identified in patients with ASD, including the previously reported splice site mutation in ASMT (IVS5+2T>C). Of the variants affecting protein sequence, only the V124I in the MTNR1B gene was absent in our comparison group. However, mutations were found in upstream regulatory regions in three of the genes investigated, ASMT, MTNR1A, and MTNR1B.
Conclusions
Our report of another ASD patient carrying the splice site mutation IVS5+2T>C, in ASMT further supports an involvement of this gene in autism. Moreover, our results also suggest that other melatonin related genes might be interesting candidates for further investigation in the search for genes involved in autism spectrum disorders and related neurobehavioral phenotypes. However, further studies of the novel variants identified in this study are warranted to shed light on their potential role in the pathophysiology of these disorders.
doi:10.1186/1755-8794-3-10
PMCID: PMC3020629  PMID: 20377855
3.  Failure to detect the 22q11.2 duplication syndrome rearrangement among patients with schizophrenia 
Chromosome aberrations have long been studied in an effort to identify susceptibility genes for schizophrenia. Chromosome 22q11.2 microdeletion is associated with DiGeorge and Velocardiofacial syndromes (DG/VCF) and provides the most convincing evidence of an association between molecular cytogenetic abnormality and schizophrenia. In addition, this region is one of the best replicated linkage findings for schizophrenia. Recently, the reciprocal microduplication on 22q11.2 has been reported as a new syndrome. Preliminary data indicates that individuals with these duplications also suffer from neuropsychiatric disorders. In this study we have investigated the appropriateness of testing schizophrenia patients for the 22q11.2 microduplication. We used multiplex ligation-dependent probe amplification (MLPA) to measure copy number changes on the 22q11.2 region in a sample of 190 patients with schizophrenia. Our results corroborate the prevalence of the 22q11.2 microdeletion in patients with schizophrenia and clinical features of DG/VCFS and do not suggest an association between 22q11.2 microduplication and schizophrenia.
doi:10.1186/1744-9081-4-10
PMCID: PMC2278148  PMID: 18284679
4.  Methylation-Specific Multiplex Ligation-Dependent Probe Amplification and Identification of Deletion Genetic Subtypes in Prader-Willi Syndrome 
Purpose: Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are complex neurodevelopmental disorders caused by loss of expression of imprinted genes from the 15q11-q13 region depending on the parent of origin. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) kits from MRC-Holland (Amsterdam, The Netherlands) were used to detect PWS and AS deletion subtypes. We report our experience with two versions of the MS-MLPA-PWS/AS kit (original A1 and newer B1) in determining methylation status and deletion subtypes in individuals with PWS. Methods: MS-MLPA analysis was performed on DNA isolated from a large cohort of PWS subjects with the MS-MLPA-PWS/AS-A1 and -B1 probe sets. Results: Both MS-MLPA kits will identify deletions in the 15q11-q13 region but the original MS-MLPA-A1 kit has a higher density of probes at the telomeric end of the 15q11-q13 region, which is more useful for identifying individuals with atypical deletions. The newer B1 kit contains more probes in the imprinting center (IC) and adjoining small noncoding RNAs useful in identifying small microdeletions. Conclusion: The A1 kit identified the typical deletions and smaller atypical deletions, whereas the B1 kit was more informative for identifying microdeletions including the IC and SNORD116 regions. Both kits should be made available for accurate characterization of PWS/AS deletion subtypes as well as evaluating for IC and SNORD116 microdeletions.
doi:10.1089/gtmb.2011.0115
PMCID: PMC3306590  PMID: 21977908
5.  Novel Submicroscopic chromosomal abnormalities detected in Autism Spectrum Disorder 
Biological psychiatry  2008;63(12):1111-1117.
Background
One genetic mechanism known to be associated with autism spectrum disorders (ASD) is chromosomal abnormalities. The identification of copy number variants (CNV) i.e. microdeletions and microduplications that are undetectable at the level of traditional cytogenetic analysis allows the potential association of submicroscopic chromosomal imbalances and human disease.
Methods
We performed array comparative genomic hybridization (aCGH) utilizing a 19K whole genome tiling path bacterial artificial chromosome (BAC) microarray on 397 unrelated subjects with autism spectrum disorder (ASD). Common CNV were excluded using a control group comprised of 372 individuals from the NIMH Genetics Initiative Control samples. Confirmation studies were performed on all remaining CNV using FISH (Fluorescence In Situ Hybridization), microsatellite analysis and/or quantitative PCR analysis.
Results
A total of 51 CNV were confirmed in 46 ASD subjects. Three maternal interstitial duplications of 15q11-q13 known to be associated with ASD were identified. The other 48 CNV ranged in size from 189 kb to 5.5 Mb and contained from 0 to ~40 RefSeq genes. Seven CNV were de novo and 44 were inherited.
Conclusions
51 autism-specific CNV were identified in 46/397 ASD patients using a 19K BAC microarray for an overall rate of 11.6%. These microdeletions and microduplications cause gene dosage imbalance in 272 genes many of which could be considered as candidate genes for autism.
doi:10.1016/j.biopsych.2008.01.009
PMCID: PMC2440346  PMID: 18374305
autism; array comparative genomic hybridization; microdeletions; microduplications
6.  Submicroscopic chromosome imbalance in patients with developmental delay and/or dysmorphism referred specifically for Fragile X testing and karyotype analysis 
Background
Microdeletion syndromes are generally identified because they usually give rise to specific phenotypic features; many of these deletions are mediated by duplicons or LCRs. The phenotypes associated with subtelomeric deletions are also becoming recognised. However, reciprocal duplication events at these loci are less easily recognised and identified, as they may give rise to milder phenotypic features, and the individuals carrying them may not therefore be referred for appropriate testing. 403 patients with developmental delay and/or dysmorphism, referred to our Genetics Centre for karyotyping and Fragile X expansion testing, were assessed for chromosome imbalance by Multiplex Ligation-dependent Probe Amplification (MLPA). Two MLPA kits were used, one containing probes for the subtelomere regions, and one containing probes for common microdeletion loci. 321 patients were tested with both kits, 75 with the subtelomere kit alone, and 7 with the microdeletion kit alone.
Results
32 patients had abnormal results; the overall abnormality detection rate was 2.5% for karyotype analysis and 7.2% for MLPA testing; 5.5% of subtelomere tests and 2.1% of microdeletion tests gave abnormal results. Of the abnormal MLPA results, 5 were in cases with cytogenetically visible abnormalities; of the remaining, submicroscopic, changes, 3 results were established as de novo and 8 were inherited; parental samples were not available for the remaining cases. None of the patients was found to have a Fragile X expansion.
Conclusion
Karyotype analysis in combination with MLPA assays for subtelomeres and microdeletion loci may be recommended for this patient group.
doi:10.1186/1755-8166-1-2
PMCID: PMC2375878  PMID: 18471307
7.  BAC array CGH in patients with Velocardiofacial syndrome-like features reveals genomic aberrations on chromosome region 1q21.1 
BMC Medical Genetics  2009;10:144.
Background
Microdeletion of the chromosome 22q11.2 region is the most common genetic aberration among patients with velocardiofacial syndrome (VCFS) but a subset of subjects do not show alterations of this chromosome region.
Methods
We analyzed 18 patients with VCFS-like features by comparative genomic hybridisation (aCGH) array and performed a face-to-face slide hybridization with two different arrays: a whole genome and a chromosome 22-specific BAC array. Putative rearrangements were confirmed by FISH and MLPA assays.
Results
One patient carried a combination of rearrangements on 1q21.1, consisting in a microduplication of 212 kb and a close microdeletion of 1.15 Mb, previously reported in patients with variable phenotypes, including mental retardation, congenital heart defects (CHD) and schizophrenia. While 326 control samples were negative for both 1q21.1 rearrangements, one of 73 patients carried the same 212-kb microduplication, reciprocal to TAR microdeletion syndrome. Also, we detected four copy number variants (CNVs) inherited from one parent (a 744-kb duplication on 10q11.22; a 160 kb duplication and deletion on 22q11.21 in two cases; and a gain of 140 kb on 22q13.2), not present in control subjects, raising the potential role of these CNVs in the VCFS-like phenotype.
Conclusions
Our results confirmed aCGH as a successful strategy in order to characterize additional submicroscopic aberrations in patients with VCF-like features that fail to show alterations in 22q11.2 region. We report a 212-kb microduplication on 1q21.1, detected in two patients, which may contribute to CHD.
doi:10.1186/1471-2350-10-144
PMCID: PMC2805625  PMID: 20030804
8.  Identification of Chromosome Abnormalities in Subtelomeric Regions Using Multiplex Ligation Dependent Probe Amplification (MLPA) Technique in 100 Iranian Patients With Idiopathic Mental Retardation 
Background
Mental retardation/Developmental delay (MR/DD) is present in 1 - 3% of the general population (1, 2). MR is defined as a significant impairment of both cognitive (IQ < 70) and social adaptive functions, with onset before 18 years of age.
Objectives
The purpose was to determine the results of subtelomeric screening by the Multiplex Ligation Dependent Probe Amplification (MLPA) Technique in 100 selected patients with idiopathic mental retardation (IMR) in Iran.
Materials and Methods
A number of 100 patients with IMR, normal karyotypes and negative fragile-X and metabolic tests were screened for subtelomeric abnormalities using MLPA technique.
Results
Nine of 100 patients showed subtelomeric abnormalities with at least one of the two MLPA kits. Deletion in a single region was found in 3 patients, and in two different subtelomeric regions in 1 patient. Duplication was only single and was present in 2 patients. Three patients were found to have both a deletion and duplication.MLPA testing in the parental samples of 7 patients which was accessible showed that 4 patients were de novo, 2 patients had inherited from a clinically normal mother, and one had inherited from a clinically normal father. Screening with the two MLPA kits (SALSA P036 and SALSA P070) proved abnormality in only five of the 9 patients.
Conclusions
So, the prevalence rate of abnormal subtelomeres using MLPA technique in patients with idiopathic MR in our study was 5 - 9%, the higher limit referring to the positive results of one of the two MLPA kits, and the lower limit representing the results of positive double-checking with the two MLPA kits.
doi:10.5812/ircmj.8221
PMCID: PMC3950786  PMID: 24693374
Ligation; Mental Retardation; Hypersomnolence Idiopathic
9.  Speech delays and behavioral problems are the predominant features in individuals with developmental delays and 16p11.2 microdeletions and microduplications 
Microdeletions and microduplications encompassing a ~593-kb region of 16p11.2 have been implicated as one of the most common genetic causes of susceptibility to autism/autism spectrum disorder (ASD). We report 45 microdeletions and 32 microduplications of 16p11.2, representing 0.78% of 9,773 individuals referred to our laboratory for microarray-based comparative genomic hybridization (aCGH) testing for neurodevelopmental and congenital anomalies. The microdeletion was de novo in 17 individuals and maternally inherited in five individuals for whom parental testing was available. Detailed histories of 18 individuals with 16p11.2 microdeletions were reviewed; all had developmental delays with below-average intelligence, and a majority had speech or language problems or delays and various behavioral problems. Of the 16 individuals old enough to be evaluated for autism, the speech/behavior profiles of seven did not suggest the need for ASD evaluation. Of the remaining nine individuals who had speech/behavior profiles that aroused clinical suspicion of ASD, five had formal evaluations, and three had PDD-NOS. Of the 19 microduplications with parental testing, five were de novo, nine were maternally inherited, and five were paternally inherited. A majority with the microduplication had delayed development and/or specific deficits in speech or language, though these features were not as consistent as seen with the microdeletions. This study, which is the largest cohort of individuals with 16p11.2 alterations reported to date, suggests that 16p11.2 microdeletions and microduplications are associated with a high frequency of cognitive, developmental, and speech delay and behavior abnormalities. Furthermore, although features associated with these alterations can be found in individuals with ASD, additional factors are likely required to lead to the development of ASD.
doi:10.1007/s11689-009-9037-4
PMCID: PMC3125720  PMID: 21731881
Array CGH; 16p11.2; Microdeletion; Microduplication; Autism; ASD
10.  Speech delays and behavioral problems are the predominant features in individuals with developmental delays and 16p11.2 microdeletions and microduplications 
Microdeletions and microduplications encompassing a ~593-kb region of 16p11.2 have been implicated as one of the most common genetic causes of susceptibility to autism/autism spectrum disorder (ASD). We report 45 microdeletions and 32 microduplications of 16p11.2, representing 0.78% of 9,773 individuals referred to our laboratory for microarray-based comparative genomic hybridization (aCGH) testing for neurodevelopmental and congenital anomalies. The microdeletion was de novo in 17 individuals and maternally inherited in five individuals for whom parental testing was available. Detailed histories of 18 individuals with 16p11.2 microdeletions were reviewed; all had developmental delays with below-average intelligence, and a majority had speech or language problems or delays and various behavioral problems. Of the 16 individuals old enough to be evaluated for autism, the speech/behavior profiles of seven did not suggest the need for ASD evaluation. Of the remaining nine individuals who had speech/behavior profiles that aroused clinical suspicion of ASD, five had formal evaluations, and three had PDD-NOS. Of the 19 microduplications with parental testing, five were de novo, nine were maternally inherited, and five were paternally inherited. A majority with the microduplication had delayed development and/or specific deficits in speech or language, though these features were not as consistent as seen with the microdeletions. This study, which is the largest cohort of individuals with 16p11.2 alterations reported to date, suggests that 16p11.2 microdeletions and microduplications are associated with a high frequency of cognitive, developmental, and speech delay and behavior abnormalities. Furthermore, although features associated with these alterations can be found in individuals with ASD, additional factors are likely required to lead to the development of ASD.
Electronic supplementary material
The online version of this article (doi:10.1007/s11689-009-9037-4) contains supplementary material, which is available to authorized users.
doi:10.1007/s11689-009-9037-4
PMCID: PMC3125720  PMID: 21731881
Array CGH; 16p11.2; Microdeletion; Microduplication; Autism; ASD
11.  A Co-segregating Microduplication of Chromosome 15q11.2 Pinpoints Two Risk Genes for Autism Spectrum Disorder 
High resolution genomic copy-number analysis has shown that inherited and de novo copy-number variations contribute significantly to autism pathology, and that identification of small chromosomal aberrations related to autism will expedite the discovery of risk genes involved. Here, we report a microduplication of chromosome 15q11.2, spanning only four genes, co-segregating with autism in a Dutch pedigree, identified by SNP microarray analysis, and independently confirmed by FISH and MLPA analysis. Quantitative RT-PCR analysis revealed over 70 % increase in peripheral blood mRNA levels for the four genes present in the duplicated region in patients, and RNA in situ hybridization on mouse embryonic and adult brain sections revealed that two of the four genes, CYFIP1 and NIPA1, were highly expressed in the developing mouse brain. These findings point towards a contribution of microduplications at chromosome 15q11.2 to autism, and highlight CYFIP1 and NIPA1 as autism risk genes functioning in axonogenesis and synaptogenesis. Thereby, these findings further implicate defects in dosage-sensitive molecular control of neuronal connectivity in autism. However, the prevalence of this microduplication in patient samples was statistically not significantly different from control samples (0.94% in patients vs 0.42% controls, p=0.247), which suggests that our findings should be interpreted with caution and indicates the need for studies that include large numbers of control subjects to ascertain the impact of these changes on a population scale.
doi:10.1002/ajmg.b.31055
PMCID: PMC2933514  PMID: 20029941
autism spectrum disorder; genetics; copy-number; gene-dosage; gene expression
12.  Mutation Screening of the PTEN Gene in Patients With Autism Spectrum Disorders and Macrocephaly 
American Journal of Medical Genetics  2007;144B(4):484-491.
Mutations in the PTEN gene are associated with a broad spectrum of disorders, including Cowden syndrome (CS), Bannayan–Riley–Ruvalcaba syndrome, Proteus syndrome, and Lhermitte–Duclos disease. In addition, PTENmutations have been described in a few patients with autism spectrum disorders (ASDs) and macrocephaly. In this study, we screened the PTEN gene for mutations and deletions in 88 patients with ASDs and macrocephaly (defined as ≥2 SD above the mean). Mutation analysis was performed by direct sequencing of all exons and flanking regions, as well as the promoter region. Dosage analysis of PTEN was carried out using multiplex ligation-dependent probe amplification (MLPA). No partial or whole gene deletions were observed. We identified a de novo missense mutation (D326N) in a highly conserved amino acid in a 5-year-old boy with autism, mental retardation, language delay, extreme macrocephaly (+9.6 SD) and polydactyly of both feet. Polydactyly has previously been described in two patients with Lhermitte–Duclos disease and CS and is thus likely to be a rare sign of PTEN mutations. Our findings suggest that PTEN mutations are a relatively infrequent cause of ASDs with macrocephaly. Screening of PTEN mutations is warranted in patients with autism and pronounced macrocephaly, even in the absence of other features of PTEN-related tumor syndromes.
doi:10.1002/ajmg.b.30493
PMCID: PMC3381648  PMID: 17427195
Cowden syndrome; Bannayan–Riley–Ruvalcaba syndrome; polydactyly; sequence analysis; multiplex ligation-dependent probe amplification
13.  Autism, language delay and mental retardation in a patient with 7q11 duplication 
Journal of Medical Genetics  2007;44(7):452-458.
Background
Chromosomal rearrangements, arising from unequal recombination between repeated sequences, are found in a subset of patients with autism. Duplications involving loci associated with behavioural disturbances especially constitute a good candidate mechanism. The Williams-Beuren Critical Region (WBCR), located in 7q11.23, is commonly deleted in the Williams-Beuren microdeletion syndrome (WBS). However, only four patients with a duplication of the WBCR have been reported so far, one with severe language delay and the three others with variable developmental, psychomotor and language delay.
Objective and Methods
In this study, we screened 206 patients with autism spectrum disorders for the WBCR duplication by quantitative microsatellite analysis and multiple ligation-dependent probe amplification (MLPA).
Results
We have identified one male patient with a de novo interstitial duplication of the entire WBCR of paternal origin. The patient had autistic disorder, severe language delay and mental retardation, with very mild dysmorphic features.
Conclusion
We report the first patient with autistic disorder who has a WBCR duplication. This observation indicates that the 7q11.23 duplication could be involved in complex clinical phenotypes, ranging from developmental or language delay to mental retardation and autism, and extends the phenotype initially reported. These findings also support the existence of one or several genes in 7q11.23 sensitive to gene dosage and involved in the development of language and social interaction.
doi:10.1136/jmg.2006.047092
PMCID: PMC1994965  PMID: 17400790
autism, mental retardation, language delay, 7q11, duplication
14.  Abnormal melatonin synthesis in autism spectrum disorders 
Molecular Psychiatry  2007;13(1):90-98.
Melatonin is produced in the dark by the pineal gland and is a key regulator of circadian and seasonal rhythms. A low melatonin level was reported in individuals with autism spectrum disorders (ASD), but the underlying cause of this deficit was unknown. The ASMT gene, encoding the last enzyme of melatonin synthesis, is located on the pseudo-autosomal region 1 of the sex chromosomes, deleted in several individuals with ASD. In this study, we sequenced all ASMT exons and promoters in individuals with ASD (n=250) and compared the allelic frequencies with controls (n=255). Non-conservative variations of ASMT were identified, including a splicing mutation present in two families with ASD, but not in controls. Two polymorphisms located in the promoter (rs4446909 and rs5989681) were more frequent in ASD compared to controls (P=0.0006) and were associated with a dramatic decrease in ASMT transcripts in blood cell lines (P=2×10−10). Biochemical analyses performed on blood platelets and/or cultured cells revealed a highly significant decrease in ASMT activity (P=2×10−12) and melatonin level (P=3×10−11) in individuals with ASD. These results indicate that a low melatonin level, caused by a primary deficit in ASMT activity, is a risk factor for ASD. They also support ASMT as a susceptibility gene for ASD and highlight the crucial role of melatonin in human cognition and behavior.
doi:10.1038/sj.mp.4002016
PMCID: PMC2199264  PMID: 17505466
autism; melatonin; circadian rhythm; sleep; HIOMT; ASMT
15.  MAPD: a probe design suite for multiplex ligation-dependent probe amplification assays 
BMC Research Notes  2010;3:137.
Background
Multiplex ligation-dependent probe amplification (MLPA) was originally described as an efficient and reliable technique for gene dosage or DNA copy number variation (CNV) analysis. Due to its low cost, reliability, sensitivity, and relative simplicity, MLPA has rapidly gained acceptance in research and diagnostic laboratories, and fills the gap between genome-wide analysis and single gene analysis. A number of new applications have been developed shortly after the introduction of MLPA, including methylation-specific MLPA (MS-MLPA), the use of MLPA in SNP genotyping, copy number analysis in segmentally duplicated regions, etc. However, probe design is time consuming and error prone. Recently software has been developed to help human genomic MLPA probe selection and optimization. For other genomes and MS-MLPA, probe design remains a challenge.
Findings
This paper describes a number of new features added to the previous H-MAPD software, which include: 1) probe selection for MS-MLPA; 2) support of mouse and rat genomes; 3) a set of new stuffer sequences. In addition, a physical-chemical property verification tool was implemented to verify user defined probes.
Conclusions
MAPD is a web-based tool which is freely available to non-commercial users. The previous H-MAPD software has been used by about 200 users from more than 30 countries. With the new features, the author hopes MAPD will bring more convenience to the MLPA community.
doi:10.1186/1756-0500-3-137
PMCID: PMC2893534  PMID: 20492694
16.  Multiplex ligation-dependent probe amplification workflow for the detection of submicroscopic chromosomal abnormalities in patients with developmental delay/intellectual disability 
Background
Array based comparative genomic hybridization (arrayCGH) has been increasingly used as the method of choice for diagnosis of patients with unexplained developmental delay/intellectual disability (DD/ID) but is not universally available for the high throughput use in routine practice. The next-generation sequencing (NGS) techniques, emerging as a new tool in clinical diagnostics, are at present quite labour-intensive and expensive. Since multiplex ligation-dependent probe amplification (MLPA) is relatively fast, easily interpreted and cost-effective, it is still a method of choice for screening large cohorts of patients with DD/ID.
Results
We prospectively studied a cohort of 150 patients with DD/ID with or without dysmorphic features or additional congenital abnormalities. We used two distinct MLPA kits, SALSA P036 and P070, for subtelomere screening and MLPA kit SALSA P245 for the 21 common microdeletion syndromes. Subtelomere analysis was performed by both kits in all patients. All imbalances were verified by follow-up MLPA kits. The MLPA analysis revealed chromosome aberrations in 21 (14%) cases: 11 subtelomeric rearrangements and 10 microdeletions.
Conclusions
We have presented the results of the investigation of patients with DD/ID obtained by using a combination of the MLPA sets for subtelomere aberrations and microdeletion syndromes followed by the confirmation of the aberrant results by the region-specific MLPA kits. The use of two subtelomeric kits per patient and investigation of all aberrations by follow-up sets has reduced the rate of false positive and negative results and improved the diagnostic yield. The relatively low cost, simplicity and reliability makes MLPA an effective first-tier cytogenetic diagnostic test for screening large cohorts of DD/ID patients.
doi:10.1186/1755-8166-6-7
PMCID: PMC3599182  PMID: 23383958
Intellectual disability; Chromosome aberrations; Genetic testing; Developing countries
17.  Exon duplications in the ATP7A gene: Frequency and Transcriptional Behaviour 
Background
Menkes disease (MD) is an X-linked, fatal neurodegenerative disorder of copper metabolism, caused by mutations in the ATP7A gene. Thirty-three Menkes patients in whom no mutation had been detected with standard diagnostic tools were screened for exon duplications in the ATP7A gene.
Methods
The ATP7A gene was screened for exon duplications using multiplex ligation-dependent probe amplification (MLPA). The expression level of ATP7A was investigated by real-time PCR and detailed analysis of the ATP7A mRNA was performed by RT-PCR followed by sequencing. In order to investigate whether the identified duplicated fragments originated from a single or from two different X-chromosomes, polymorphic markers located in the duplicated fragments were analyzed.
Results
Partial ATP7A gene duplication was identified in 20 unrelated patients including one patient with Occipital Horn Syndrome (OHS). Duplications in the ATP7A gene are estimated from our material to be the disease causing mutation in 4% of the Menkes disease patients. The duplicated regions consist of between 2 and 15 exons. In at least one of the cases, the duplication was due to an intra-chromosomal event. Characterization of the ATP7A mRNA transcripts in 11 patients revealed that the duplications were organized in tandem, in a head to tail direction. The reading frame was disrupted in all 11 cases. Small amounts of wild-type transcript were found in all patients as a result of exon-skipping events occurring in the duplicated regions. In the OHS patient with a duplication of exon 3 and 4, the duplicated out-of-frame transcript coexists with an almost equally represented wild-type transcript, presumably leading to the milder phenotype.
Conclusions
In general, patients with duplication of only 2 exons exhibit a milder phenotype as compared to patients with duplication of more than 2 exons. This study provides insight into exon duplications in the ATP7A gene.
doi:10.1186/1750-1172-6-73
PMCID: PMC3240829  PMID: 22074552
18.  Sequencing ASMT Identifies Rare Mutations in Chinese Han Patients with Autism 
PLoS ONE  2013;8(1):e53727.
Melatonin is involved in the regulation of circadian and seasonal rhythms and immune function. Prior research reported low melatonin levels in autism spectrum disorders (ASD). ASMT located in pseudo-autosomal region 1 encodes the last enzyme of the melatonin biosynthesis pathway. A previous study reported an association between ASD and single nucleotide polymorphisms (SNPs) rs4446909 and rs5989681 located in the promoter of ASMT. Furthermore, rare deleterious mutations were identified in a subset of patients. To investigate the association between ASMT and autism, we sequenced all ASMT exons and its neighboring region in 398 Chinese Han individuals with autism and 437 healthy controls. Although our study did not detect significant differences of genotypic distribution and allele frequencies of the common SNPs in ASMT between patients with autism and healthy controls, we identified new rare coding mutations of ASMT. Among these rare variants, 4 were exclusively detected in patients with autism including a stop mutation (p.R115W, p.V166I, p.V179G, and p.W257X). These four coding variants were observed in 6 of 398 (1.51%) patients with autism and none in 437 controls (Chi-Square test, Continuity Correction p = 0.032, two-sided). Functional prediction of impact of amino acid showed that p.R115W might affect protein function. These results indicate that ASMT might be a susceptibility gene for autism. Further studies in larger samples are needed to better understand the degree of variation in this gene as well as to understand the biochemical and clinical impacts of ASMT/melatonin deficiency.
doi:10.1371/journal.pone.0053727
PMCID: PMC3547942  PMID: 23349736
19.  Variations of the Candidate SEZ6L2 Gene on Chromosome 16p11.2 in Patients with Autism Spectrum Disorders and in Human Populations 
PLoS ONE  2011;6(3):e17289.
Background
Autism spectrum disorders (ASD) are a group of severe childhood neurodevelopmental disorders with still unknown etiology. One of the most frequently reported associations is the presence of recurrent de novo or inherited microdeletions and microduplications on chromosome 16p11.2. The analysis of rare variations of 8 candidate genes among the 27 genes located in this region suggested SEZ6L2 as a compelling candidate.
Methodology/Principal Findings
We further explored the role of SEZ6L2 variations by screening its coding part in a group of 452 individuals, including 170 patients with ASD and 282 individuals from different ethnic backgrounds of the Human Genome Diversity Panel (HGDP), complementing the previously reported screening. We detected 7 previously unidentified non-synonymous variations of SEZ6L2 in ASD patients. We also identified 6 non-synonymous variations present only in HGDP. When we merged our results with the previously published, no enrichment of non-synonymous variation in SEZ6L2 was observed in the ASD group compared with controls.
Conclusions/Significance
Our results provide an extensive ascertainment of the genetic variability of SEZ6L2 in human populations and do not support a major role for SEZ6L2 sequence variations in the susceptibility to ASD.
doi:10.1371/journal.pone.0017289
PMCID: PMC3048866  PMID: 21394203
20.  Utility of MLPA in mutation analysis and carrier detection for Duchenne muscular dystrophy 
CONTEXT:
Multiplex ligation probe amplification (MLPA) is a new technique to identify deletions and duplications and can evaluate all 79 exons in dystrophin gene in patients with Duchenne muscular dystrophy (DMD). Being semi-quantitative, MLPA is also effective in detecting duplications and carrier testing of females; both of which cannot be done using multiplex PCR. It has found applications in diagnostics of many genetic disorders.
AIM:
To study the utility of MLPA in diagnosis and carrier detection for DMD.
MATERIALS AND METHODS:
Mutation analysis and carrier detection was done by multiplex PCR and MLPA and the results were compared.
RESULTS AND CONCLUSIONS:
We present data showing utility of MLPA in identifying mutations in cases with DMD/BMD. In the present study using MLPA, we identified mutations in additional 5.6% cases of DMD in whom multiplex PCR was not able to detect intragenic deletions. In addition, MLPA also correctly confirmed carrier status of two obligate carriers and revealed carrier status in 6 of 8 mothers of sporadic cases.
doi:10.4103/0971-6866.96667
PMCID: PMC3385188  PMID: 22754229
Dystrophin; Duchenne and Becker muscular dystrophies; multiplex ligation-dependent probe amplification; carrier detection
21.  Rapid screening for chromosomal aneuploidies using array-MLPA 
BMC Medical Genetics  2011;12:68.
Background
Chromosome abnormalities, especially trisomy of chromosome 21, 13, or 18 as well as sex chromosome aneuploidy, are a well-established cause of pregnancy loss. Cultured cell karyotype analysis and FISH have been considered reliable detectors of fetal abnormality. However, results are usually not available for 3-4 days or more. Multiplex ligation-dependent probe amplification (MLPA) has emerged as an alternative rapid technique for detection of chromosome aneuploidies. However, conventional MLPA does not allow for relative quantification of more than 50 different target sequences in one reaction and does not detect mosaic trisomy. A multiplexed MLPA with more sensitive detection would be useful for fetal genetic screening.
Methods
We developed a method of array-based MLPA to rapidly screen for common aneuploidies. We designed 116 universal tag-probes covering chromosomes 13, 18, 21, X, and Y, and 8 control autosomal genes. We performed MLPA and hybridized the products on a 4-well flow-through microarray system. We determined chromosome copy numbers by analyzing the relative signals of the chromosome-specific probes.
Results
In a blind study of 161 peripheral blood and 12 amniotic fluid samples previously karyotyped, 169 of 173 (97.7%) including all the amniotic fluid samples were correctly identified by array-MLPA. Furthermore, we detected two chromosome X monosomy mosaic cases in which the mosaism rates estimated by array-MLPA were basically consistent with the results from karyotyping. Additionally, we identified five Y chromosome abnormalities in which G-banding could not distinguish their origins for four of the five cases.
Conclusions
Our study demonstrates the successful application and strong potential of array-MLPA in clinical diagnosis and prenatal testing for rapid and sensitive chromosomal aneuploidy screening. Furthermore, we have developed a simple and rapid procedure for screening copy numbers on chromosomes 13, 18, 21, X, and Y using array-MLPA.
doi:10.1186/1471-2350-12-68
PMCID: PMC3111339  PMID: 21575262
22.  Genetic Analysis of Dystrophin Gene for Affected Male and Female Carriers with Duchenne/Becker Muscular Dystrophy in Korea 
Journal of Korean Medical Science  2012;27(3):274-280.
Duchenne and Becker muscular dystrophy (DMD/BMD) are X-linked recessive disorders caused by mutation in dystrophin gene. We analyzed the results of a genetic test in 29 DMD/BMD patients, their six female relatives, and two myopathic female patients in Korea. As the methods developed, we applied different procedures for dystrophin gene analysis; initially, multiplex polymerase chain reaction was used, followed by multiplex ligation-dependent probe amplification (MLPA). Additionally, we used direct DNA sequencing for some patients who had negative results using the above methods. The overall mutation detection rate was 72.4% (21/29) in DMD/BMD patients, identifying deletions in 58.6% (17/29). Most of the deletions were confined to the central hot spot region between exons 44 and 55 (52.9%, 7/19). The percentage of deletions and duplications revealed by MLPA was 45.5% (5/11) and 27.2% (3/11), respectively. Using the MLPA method, we detected mutations confirming their carrier status in all female relatives and symptomatic female patients. In one patient in whom MLPA revealed a single exon deletion of the dystrophin gene, subsequent DNA sequencing analysis identified a novel nonsense mutation (c.4558G > T; Gln1520X). The MLPA assay is a useful quantitative method for detecting mutation in asymptomatic or symptomatic carriers as well as DMD/BMD patients.
doi:10.3346/jkms.2012.27.3.274
PMCID: PMC3286774  PMID: 22379338
Gene Amplification; Duchenne/Becker Muscular Dystrophy; Deletion; Duplication
23.  Mutation analysis of the SLC26A4, FOXI1 and KCNJ10 genes in individuals with congenital hearing loss 
PeerJ  2014;2:e384.
Pendred syndrome (PDS) and DFNB4 comprise a phenotypic spectrum of sensorineural hearing loss disorders that typically result from biallelic mutations of the SLC26A4 gene. Although PDS and DFNB4 are recessively inherited, sequencing of the coding regions and splice sites of SLC26A4 in individuals suspected to be affected with these conditions often fails to identify two mutations. We investigated the potential contribution of large SLC26A4 deletions and duplications to sensorineural hearing loss (SNHL) by screening 107 probands with one known SLC26A4 mutation by Multiplex Ligation-dependent Probe Amplification (MLPA). A heterozygous deletion, spanning exons 4–6, was detected in only one individual, accounting for approximately 1% of the missing mutations in our cohort. This low frequency is consistent with previously published MLPA results. We also examined the potential involvement of digenic inheritance in PDS/DFNB4 by sequencing the coding regions of FOXI1 and KCNJ10. Of the 29 probands who were sequenced, three carried nonsynonymous variants including one novel sequence change in FOXI1 and two polymorphisms in KCNJ10. We performed a review of prior studies and, in conjunction with our current data, conclude that the frequency of FOXI1 (1.4%) and KCNJ10 (3.6%) variants in PDS/DFNB4 individuals is low. Our results, in combination with previously published reports, indicate that large SLC26A4 deletions and duplications as well as mutations of FOXI1 and KCNJ10 play limited roles in the pathogenesis of SNHL and suggest that other genetic factors likely contribute to the phenotype.
doi:10.7717/peerj.384
PMCID: PMC4017815  PMID: 24860705
Pendred; MLPA; DFNB4; SLC26A4; FOXI1 and KCNJ10; Genotyping; Genetics; SNHL
24.  Mutation screening of ASMT, the last enzyme of the melatonin pathway, in a large sample of patients with Intellectual Disability 
BMC Medical Genetics  2011;12:17.
Background
Intellectual disability (ID) is frequently associated with sleep disorders. Treatment with melatonin demonstrated efficacy, suggesting that, at least in a subgroup of patients, the endogenous melatonin level may not be sufficient to adequately set the sleep-wake cycles. Mutations in ASMT gene, coding the last enzyme of the melatonin pathway have been reported as a risk factor for autism spectrum disorders (ASD), which are often comorbid with ID. Thus the aim of the study was to ascertain the genetic variability of ASMT in a large cohort of patients with ID and controls.
Methods
Here, we sequenced all exons of ASMT in a sample of 361 patients with ID and 440 controls. We then measured the ASMT activity in B lymphoblastoid cell lines (BLCL) of patients with ID carrying an ASMT variant and compared it to controls.
Results
We could identify eleven variations modifying the protein sequence of ASMT (ID only: N13H, N17K, V171M, E288D; controls only: E61Q, D210G, K219R, P243L, C273S, R291Q; ID and controls: L298F) and two deleterious splice site mutations (IVS5+2T>C and IVS7+1G>T) only observed in patients with ID. We then ascertained ASMT activity in B lymphoblastoid cell lines from patients carrying the mutations and showed significantly lower enzyme activity in patients carrying mutations compared to controls (p = 0.004).
Conclusions
We could identify patients with deleterious ASMT mutations as well as decreased ASMT activity. However, this study does not support ASMT as a causative gene for ID since we observed no significant enrichment in the frequency of ASMT variants in ID compared to controls. Nevertheless, given the impact of sleep difficulties in patients with ID, melatonin supplementation might be of great benefit for a subgroup of patients with low melatonin synthesis.
doi:10.1186/1471-2350-12-17
PMCID: PMC3034665  PMID: 21251267
25.  High Frequency of BMPR2 Exonic Deletions/Duplications in Familial Pulmonary Arterial Hypertension 
Rationale: Previous studies have shown that approximately 55% of patients with familial pulmonary arterial hypertension (FPAH) have BMPR2 coding sequence mutations. However, direct sequencing does not detect other types of heterozygous mutations, such as exonic deletions/duplications.
Objective: To estimate the frequency of BMPR2 exonic deletions/duplications in FPAH.
Methods: BMPR2 mRNA from lymphoblastoid cell lines of 30 families with PAH and 14 patients with idiopathic PAH (IPAH) was subjected to reverse transcriptase–polymerase chain reaction (RT-PCR) and sequencing. Sequencing of genomic DNA was used to identify point mutations in splice donor/acceptor sites. Multiplex ligation-dependent probe amplification (MLPA) was used to detect exonic deletions/duplications with verification by real-time PCR.
Measurements and Main Results: Eleven (37%) patients with FPAH had abnormally sized RT-PCR products. Four of the 11 patients were found to have splice-site mutations resulting in aberrant splicing, and exonic deletions/duplications were detected in the remaining seven patients. MLPA identified three deletions/duplications that were not detectable by RT-PCR. Coding sequence point mutations were identified in 11 of 30 (37%) patients. Mutations were identified in 21 of 30 (70%) patients with FPAH, with 10 of 21 mutations (48%) being exonic deletions/duplications. Two of 14 (14%) patients with IPAH exhibited BMPR2 point mutations, whereas none showed exonic deletions/duplications.
Conclusions: Our study indicates that BMPR2 exonic deletions/duplications in patients with FPAH account for a significant proportion of mutations (48%) that until now have not been screened for. Because the complementary approach used in this study is rapid and cost effective, screening for BMPR2 deletions/duplications by MLPA and real-time PCR should accompany direct sequencing in all PAH testing.
doi:10.1164/rccm.200602-165OC
PMCID: PMC2648061  PMID: 16728714
dosage; genetics; multiplex ligation-dependent probe amplification

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