Large Heterozygous Deletion Research Articles

A compound heterozygous missense mutation and a large deletion in the KCTD7 gene presenting as an opsoclonus-myoclonus ataxia-like syndrome

Mutations in the potassium channel-related gene KCTD7 were described so far in a single family with progressive myoclonus epilepsy. We describe a unique phenotype: acute onset of myoclonus and ataxia, associated with abnormal opsoclonus-like eye movements; improvement of clinical symptoms under steroid treatment; and appearance of epileptic activity on EEG 2 years later without overt seizures. After excluding possible genetic causes, whole-genome exome sequencing was performed in order to identify the causative gene. One heterozygous missense mutation (R84W) was detected by exome sequencing and a large heterozygous deletion of exons 3 and 4 by MLPA analysis. The father is heterozygous for the R84W mutation and the mother is heterozygous for the exon 3+4 deletion. The mutation affects a highly conserved segment of the predicted protein, changing a basic amino acid into neutral. The large deletion probably results in a truncated protein. The different phenotype broadens the spectrum of KCTD7-related diseases. Therefore, patients diagnosed as having opsoclonus-myoclonus with an atypical course should be evaluated for KCTD7 mutations.

Detection of a large heterozygous deletion and a splicing defect in the CFTR transcripts from nasal swab of a Japanese case of cystic fibrosis

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in CFTR (CF transmembrane conductance regulator). Although CF is the most common hereditary disease in Caucasians, it is rare in Asian populations. Common disease-causing mutations of CFTR in Caucasians are rarely identified in Japanese patients with CF. In the present study, CFTR transcripts from nasal swab were analyzed in a Japanese boy, in addition to conventional PCR and direct sequence of all exons, their boundaries and promoter region of the CFTR gene. The boy was diagnosed with CF by chronic respiratory infection and the elevated sweat chloride level. None of the disease-causing mutations of CFTR was detected by the conventional analysis. Cloning and sequence of the CFTR transcripts revealed a heterozygous deletion spanning exons 16, 17a and 17b. The deletion was confirmed by multiplex ligation-dependent probe amplification and the direct sequence of the junction fragment obtained from the genomic DNA by primer walking, which revealed the mutation c.2908+1085_3367+260del7201. We also identified a splicing defect: deletion/skipping of exon 1 in the CFTR transcript from the other allele. The analysis of CFTR transcripts from nasal swab is recommended in the genetic analysis of CF in Japanese.

Targeted array CGH as a valuable molecular diagnostic approach: Experience in the diagnosis of mitochondrial and metabolic disorders

Oligonucleotide array-based comparative genomic hybridization (aCGH) targeted to coding exons of genes of interest has been proven to be a valuable diagnostic tool to complement with Sanger sequencing for the detection of large deletions/duplications. We have developed a custom designed oligonucleotide aCGH platform for this purpose. This array platform provides tiled coverage of the entire mitochondrial genome and high-density coverage of a set of nuclear genes involving mitochondrial and metabolic disorders and can be used to evaluate large deletions in targeted genes. A total of 1280 DNA samples from patients suspected of having mitochondrial or metabolic disorders were evaluated using this targeted aCGH. We detected 40 (3%) pathogenic large deletions in unrelated individuals, including 6 in genes responsible for mitochondrial DNA (mtDNA) depletion syndromes, 23 in urea cycle genes, 11 in metabolic and related genes. Deletion breakpoints have been confirmed in 31 cases by PCR and sequencing. The possible deletion mechanism has been discussed. These results illustrate the successful utilization of targeted aCGH to detect large deletions in nuclear and mitochondrial genomes. This technology is particularly useful as a complementary diagnostic test in the context of a recessive disease when only one mutant allele is found by sequencing. For female carriers of X-linked disorders, if sequencing analysis does not detect point mutations, targeted aCGH should be considered for the detection of large heterozygous deletions.

Revisiting the molecular epidemiology of factor XI deficiency: Nine new mutations and an original large 4qTer deletion in western Brittany (France)

Constitutional deficiency in factor XI (FXI) is a rare bleeding disorder in the general population, with the exception of Ashkenazi Jews. During the last decade, the detection of FXI-deficient patients has shifted from clinical screening identifying mostly severe bleeders to biological screening combining findings of prolonged activated partial thromboplastin time and FXI coagulation activity (FXI:C) below 50 U/dl. The goal of this study was to determine the molecular basis of FXI deficiency in western Brittany, France. Over the course of four years, we detected 98 FXI-deficient patients through biological screening, and 44 patients agreed to participate in this study corresponding to 25 index cases. We developed an efficient mutation detection strategy (combining direct sequencing and QFM-PCR to search for heterozygous rearrangements in a routine setting) that detected F11 mutations in 24 out of the 25 index cases. An unexpected allelic heterogeneity was found, with 14 different single point mutations being detected, among which nine are new. Moreover, a large heterozygous deletion of the entire F11 gene was detected, and was then further defined using a CGH array as a 4q34.2 telomeric deletion of 7 Mb containing 77 genes. We propose that the observed recurrent mutations may be considered as genetic tags of a population. This study highlights the importance of screening for large deletions in molecular studies of F11 .

A large heterozygous deletion including the entire C1 inhibitor gene in a sporadic case of hereditary angio-oedema

C1 inhibitor (C1-INH) deficiency [hereditary or acquired angio-oedema (HAE or AAE)] is characterized by recurring episodes of subcutaneous or submucosal oedema. Many different mutations in the C1-INH gene have been identified as a cause of HAE. We investigated the molecular basis of the disease in a Japanese woman with sporadic HAE. Direct sequencing of genomic DNA revealed no point mutation in the C1-INH gene. Quantitative real-time PCR showed that the copy number of the C1-INH gene in the patient was half that of a healthy control. Furthermore, we identified a 650-kbp deletion on the chromosome, which included the C1-INH gene. We evaluated the correlation between the patient's attacks and her coagulation activity. The levels of D-dimer were high during the angio-oedema attacks, and often exceeded the normal range even during remission, thus the level of D-dimer reflected the activity of HAE in this patient.

Abstract 5599: Screening for large genomic rearrangements in the FANCA and FANCJ genes reveals extensive genomic FANCA deletion in a Finnish breast cancer family

Abstract A portion of familial breast cancer is accounted for by mutations in the same genes that are inactivated in Fanconi anemia (FA). In the current study we set out to evaluate the role of large genomic rearrangements in the FANCA and FANCJ/BRIP1 genes in Finnish breast cancer families. Mutations in the upstream FANCA gene account for the majority of FA patients, and about one-third of all FANCA mutations are large intragenic deletions. Based on the gene's hypermutable nature, the protein's interaction with BRCA1 and its involvement in genomic integrity maintenance, we considered FANCA as a candidate for breast cancer susceptibility. In FANCJ, which is a downstream FA and known breast cancer susceptibility gene, early truncating mutations have been reported, but no large deletions have been recognized so far. For the present study, blood DNA from affected index persons of 100 Northern Finnish breast cancer families were assessed for possible constitutional exonic deletions or amplifications in FANCA and FANCJ by using the multiplex ligation-dependent probe amplification (MLPA) method. We identified a novel large heterozygous FANCA deletion in one of the studied breast cancer families, removing the promoter region and the first 12 exons of the gene. The defective allele was absent from all of the tested 124 controls as well as 540 unselected breast cancer cases, but was present in the father of the index patient having pancreatic cancer. No large deletions in FANCJ were uncovered. We conclude that large FANCA deletions might contribute to breast cancer susceptibility, therefore more extensive studies on their involvement in the disease are needed. To our knowledge, this is the first report on the existence of a large-size genomic deletion in an upstream FA gene in association with familial breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5599. doi:10.1158/1538-7445.AM2011-5599

Look for the silent when other tests cannot speak (a case of unexpected rare thalassaemia)

Microcytic anaemia is a common consultation in haematology practice. The common causes are iron deficiency, thalassaemia or anaemia of chronic disease. The diagnosis can be directly obtained through appropriate laboratory tests. However, in the absence of a positive history or a laboratory test, the diagnosis may be obscure and more sophisticated laboratory tests may be required. A 27-year-old Australian woman from a Scottish-Welsh ancestry was referred with long standing microcytic anaemia. The laboratory tests including iron studies, haemoglobin HPLC and electro-phoresis were normal and repeated twice to exclude pre-analytical and analytical variability. Other causes of microcytic anaemia including lead toxicity, copper or zinc deficiency, chronic inflammatory disease or sideroblastic anaemia were excluded. The patient’s blood sample was sent to the Royal Prince Alfred hospital for molecular testing for possible underlying thalassaemia. A heterozygous large deletion in the beta globin region with an intact alpha globin using DNA sequencing and multiplex ligation-dependent probe amplification (MLPA) was detected. The finding supports the diagnosis of epsilon-gamma-delta-beta (eγδβ)o thalassaemia. This type of normal HbA2 beta thalassaemia or silent beta thalassaemia has been previously reported in a family with Scottish-Irish ethnic background in NSW. 1

Large genomic deletions in CACNA1A cause episodic ataxia type 2

Episodic ataxia (EA) syndromes are heritable diseases characterized by dramatic episodes of imbalance and incoordination. EA type 2 (EA2), the most common and the best characterized subtype, is caused by mostly nonsense, splice site, small indel, and sometimes missense mutations in CACNA1A. Direct sequencing of CACNA1A fails to identify mutations in some patients with EA2-like features, possibly due to incomplete interrogation of CACNA1A or defects in other EA genes not yet defined. Previous reports described genomic deletions between 4 and 40 kb in EA2. In 47 subjects with EA (26 with EA2-like features) who tested negative for mutations in the known EA genes, we used multiplex ligation-dependent probe amplification to analyze CACNA1A for exonic copy number variations. Breakpoints were further defined by long-range PCR. We identified distinct multi-exonic deletions in three probands with classic EA2-like features: episodes of prolonged vertigo and ataxia triggered by stress and fatigue, interictal nystagmus, with onset during infancy or early childhood. The breakpoints in all three probands are located in Alu sequences, indicating errors in homologous recombination of Alu sequences as the underlying mechanism. The smallest deletion spanned exons 39 and 40, while the largest deletion spanned 200 kb, missing all but the first three exons. One deletion involving exons 39 through 47 arose spontaneously. The search for mutations in CACNA1A appears most fruitful in EA patients with interictal nystagmus and onset early in life. The finding of large heterozygous deletions suggests haploinsufficiency as a possible pathomechanism of EA2.

PPARγ is functionally expressed in clear cell renal cell carcinoma

Peroxisome proliferator-activated receptor gamma (PPARγ) agonists have been demonstrated to exert an inhibitory effect on cell growth in several tumor models, including clear cell renal cell carcinoma (CCRCC). PPARγ has therefore been proposed to be a potential therapeutic target. Thus, the PPARγ gene must be expressed and not altered in cancer cells. We have therefore analyzed tumor specimens collected from 63 patients with CCRCC who underwent partial or total nephrectomy. The multiplex ligation-dependent probe amplification (MLPA) assay was used to detect deletions in the PPARγ gene. The majority of the tumors (48/63; 76.2%) did not present alterations. Two samples (3.2%) presented a deletion of the non-coding exon A1. Nine samples (14.3%) showed large heterozygous deletions in chromosome 3p including PPARγ. Potential mutations were analyzed by DNA sequencing of the 6 coding exons of the PPARγ gene. No mutation was found in exons 1-5. In exon 6, a silent polymorphism was detected in 14 samples (22.2%). CCRCC were found to express the PPARγ1 isoform. The expression level of PPARγ was measured by real-time quantitative PCR. A significantly reduced transcript level was associated with an elevated Fuhrman grade. Finally, we analyzed the expression of angiopoietin-like 4, a known PPARγ target gene, in CCRCC cell lines cultured in the presence of rosiglitazone, a PPARγ agonist. A strong induction was found in the 3 cell lines tested, indicating that PPARγ is functional in all these cell lines. In conclusion, we show here that PPARγ is expressed and functional in CCRCC, prerequisites for being a potential target for CCRCC treatment.

High-Resolution genomic arrays identify CNVs that phenocopy the chromosome 22q11.2 deletion syndrome

The 22q11 Deletion Syndrome includes the overlapping phenotypes of DiGeorge/Velocardiofacial Syndromes, characterized by conotruncal heart defects, cleft palate, thymus, and parathyroid gland dysplasia. The majority (90%) of patients harbor detectable chr22q11.2 deletions, but a genetic etiology for the remainder of patients without a deletion can remain undefined despite major birth defects. We analyzed DNA from eight patients with normal 22q11 FISH studies by high-density single nucleotide polymorphism (SNP) arrays and identified potentially pathogenic copy number variants (CNVs) in four of eight patients. Two patients showed large CNVs in regions of known genomic disorders: one a deletion of distal chr22q11.2 and the other a duplication of chr5q35. A 3-Mb deletion of chr19p13.3 that includes a gene associated with conotruncal heart defects was found in a third patient. Two potentially pathogenic CNVs were found in a fourth patient: a large heterozygous deletion of chr6p24 and a smaller duplication of chr9p24. Our findings support a recent consensus statement advocating chromosomal microarray analysis as a first-line diagnostic approach for patients with multiple congenital anomalies. In patients with phenotypes suggestive of the 22q11.2 syndrome spectrum and normal FISH, microarray analysis can uncover the molecular basis of other genomic disorders whose features overlap those of 22q11.2 deletions.

Large SGCE deletion contributes to Taiwanese myoclonus–dystonia syndrome

We report three novel deletions of the SGCE gene in three families with myoclonus–dystonia (M–D) syndrome in Taiwan. Their clinical characteristics included: early onset, dominant myoclonus and dystonia in the neck, trunk and upper limbs. By direct sequencing of the SGCE gene coding regions, we identified a small heterozygous deletion (c.842delA) in exon 7 of the three sibs and asymptomatic father in the first family and an eight-base heterozygous deletion (c.524_531del) in exon 5 of the mother and a daughter in the second family. Using multiple ligation-dependent probe amplification (MLPA), a large heterozygous deletion of 2–11 exons was identified in the father and a son in the third family which was undetected by initial sequencing. It is the largest intragenic deletion ever reported. In conclusion, we have identified three novel mutations of SGCE in the respective three M–D families. The large deletion was responsible for one third of these M–D families which might implicate an important contribution to Taiwanese M–D syndrome. We suggest that the contribution of large deletion should be further verified in a large cohort of patients with M–D syndrome in Han Chinese.

Application of oligonucleotide array CGH in the detection of a large intragenic deletion in POLG associated with Alpers Syndrome

Mutations in the polymerase γ ( POLG) gene are among the most common causes of mitochondrial disease and more than 160 POLG mutations have been reported. However, a large proportion of patients suspected of having POLG mutations only have one (heterozygous) definitive pathogenic mutation identified. Using oligonucleotide array CGH, we identified a compound heterozygous large intragenic deletion encompassing exons 15–21 of this gene in a child with Alpers syndrome due to mtDNA depletion. This is the first large POLG deletion reported and the findings show the clinical utility of using array CGH in cases where a single heterozygous mutation has been identified in POLG.

High frequency of multiexonic deletion of the GCH1 gene in a Taiwanese cohort of dopa‐response dystonia

Large deletions in the GCH1 gene have been reported in a minority of cases of dopa-responsive dystonia (DRD). In this study, we performed an extensive clinical and genetic investigation of 22 affected members in eight families. Sequence analysis revealed five different mutations in five families (n = 10); Ser81Pro (novel), Ser76X, Gly203Arg, 249del A, and IVS5 + 3insT. Applying multiple ligation-dependent probe amplification analysis, we detected a large heterozygous deletion of exons 1-3 in the remaining three families (n = 12), which was verified by quantitative real-time PCR analysis. Therefore, the large deletion accounted for 37.5% of the total families and 55% of our DRD population. The deletion appeared to have high penetrance and was associated with multifocal dystonia and adult onset in males. Adult-onset patients were commonly presenting with resting tremor, rigidity, and bradykinesia, indistinguishable from those in Parkinson's disease. In conclusion, a high frequency of multiexonic deletion of GCH1 was identified in the Taiwanese DRD population. By dosage analysis, we were able to detect a mutation in all patients. Our study demonstrates that dosage analysis is necessary for molecular diagnostics in DRD patients of Han Chinese ethnicity.

Screening of autosomal gene deletions in patients with hypogonadotropic hypogonadism using multiplex ligation‐dependent probe amplification: detection of a hemizygosis for the fibroblast growth factor receptor 1

Congenital hypogonadotropic hypogonadism with anosmia (Kallmann syndrome) or with normal sense of smell is a heterogeneous genetic disorder caused by defects in the synthesis, secretion and action of gonadotrophin-releasing hormone (GnRH). Mutations involving autosomal genes have been identified in approximately 30% of all cases of hypogonadotropic hypogonadism. However, most studies that screened patients with hypogonadotropic hypogonadism for gene mutations did not include gene dosage methodologies. Therefore, it remains to be determined whether patients without detected point mutation carried a heterozygous deletion of one or more exons. We used the multiplex ligation-dependent probe amplification (MLPA) assay to evaluate the potential contribution of heterozygous deletions of FGFR1, GnRH1, GnRHR, GPR54 and NELF genes in the aetiology of GnRH deficiency. We studied a mutation-negative cohort of 135 patients, 80 with Kallmann syndrome and 55 with normosmic hypogonadotropic hypogonadism. One large heterozygous deletion involving all FGFR1 exons was identified in a female patient with sporadic normosmic hypogonadotropic hypogonadism and mild dimorphisms as ogival palate and cavus foot. FGFR1 hemizygosity was confirmed by gene dosage with comparative multiplex and real-time PCRs. FGFR1 or other autosomal gene deletion is a possible but very rare event and does not account for a significant number of sporadic or inherited cases of isolated GnRH deficiency.

A Large Deletion in the Succinate Dehydrogenase B Gene (SDHB) in a Japanese Patient with Abdominal Paraganglioma and Concomitant Metastasis

Recently, mutations in nuclear genes encoding two mitochondrial complex II subunit proteins, Succinate dehydrogenase D (SDHD) and SDHB, have been found to be associated with the development of familial pheochromocytomas and paragangliomas (hereditary pheochromocytoma/paraganglioma syndrome: HPPS). Growing evidence suggests that the mutation of SDHB is highly associated with abdominal paraganglioma and the following distant metastasis (malignant paraganglioma). In the present study, we used multiplex ligation dependent probe amplification (MLPA) analysis to identify a large heterozygous SDHB gene deletion encompassing sequences corresponding to the promoter region, in addition to exon 1 and exon 2 malignant paraganglioma patient in whom previously characterized SDHB mutations were undetectable. This is the first Japanese case report of malignant paraganglioma, with a large SDHB deletions. Our present findings strongly support the notion that large deletions in the SDHB gene should be considered in patients lacking characterized SDHB mutations.

Genotyping of CYP21A2 for Congenital Adrenal Hyperplasia Screening using Allele-Specific Primer Extension followed by Bead Array Hybridization

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease caused by mutations in the CYP21A2 gene, which codes for steroid 21-hydroxylase. More than 90% of patients with CAH have mutations in CYP21A2 or have large deletions in the RCCX module on chromosome 6p21.3, which also includes the pseudogene CYP21A1P. Genotyping of CYP21A2 is required for diagnosis of CAH, but current genotyping methods, such as direct sequencing, allele-specific PCR amplification, or PCR amplification and restriction fragment length polymorphism (PCR-RFLP) still need further improvements to reduce test time and cost. We developed a novel CAH mutation screening method based on allele-specific primer extension (ASPE), followed by bead-array hybridization, for the ten major point mutation sites and the 8 bp deletion in CYP21A2, and a long PCR assay to detect large deletions between CYP21A1P and CYP21A2. After the first long PCR amplification, a second short PCR amplification was adapted to increase the ASPE efficiency. The total genotyping procedure takes approximately 8 hours. Eighteen CAH patients and two controls were tested using the bead-array method. Homozygous or heterozygous large gene deletions and three point mutation sites were detected by this method, and most of the results were consistent with sequencing or PCR-RFLP analysis. Nine of the 18 patients had a large deletion in the RCCX module, which was not easily detected using the conventional genotyping method. A novel CAH mutation screening method has been developed to detect ten point mutations and the 8 bp deletion in CYP21A2, as well as large deletions between CYP21A1P and CYP21A2. This novel genotyping strategy is superior to PCR-RFLP-based methods and equally as accurate as sequencing.

The Use of High Resolution Melting (HRM) Analysis for Molecular Gene Defects of Type 3 Von Willebrand Disease: Studies of An Italian Cohort of 10 Patients.

Abstract 3498Poster Board III-435 IntroductionType 3 von Willebrand disease (VWD3) is a severe autosomal recessive inherited bleeding disorder caused by a virtually complete absence of von Willebrand Factor (VWF). Classically, patients are homozygous or compound heterozygous for null alleles due to nonsense mutations, small insertions/deletions, splice site defects or, more rarely, large gene deletions spread throughout the VWF gene. Nevertheless, several missense mutations have also been reported. Aims of the study, patients and methodsThe aim of this study was to investigate the molecular basis of VWD3 in 10 Italian patients using DNA direct sequencing, High Resolution Melting (HRM) analysis and duplex PCR. HRM is a simple, low-cost, and rapid PCR-based method for detecting sequence variation by measuring changes in the melting temperature of double stranded DNA. Duplex PCR was used to screen for the presence of some known large deletions causing VWD3: the 61-kb deletion encompassing exons 6-16 (Xie et al. Blood Cells Mol Dis. 2006; 36: 385), the 253-kb deletion involving the whole VWF gene (Schneppenheim et al. J Thromb Haemost. 2007; 5: 722), the exons 1-3 deletion (Mohl et al. J Thromb Haemost. 2008; 6: 1729), and the exons 4-5 deletion (Sutherland et al. Blood. 2009; 114: 1091). Results and discussionTwenty-four exons were analyzed by direct sequencing, 21 exons by HRM and, so far, 6 exons using both methods. The following mutations were identified in 8 of the 10 patients investigated: 2157delA/7729+7C>T; C2184S*/undetermined; Q1526X*/C2325S*; del ex1-3/3940delG*; 8155+1G>T*/8155+1G>T*; E1549X*/undetermined; 658-2A>G*/658-2A>G*; del ex 1-3/undetermined. Direct sequencing revealed 7 mutations, HRM analysis could detect 2 defects (2157delA, C2325S) and duplex PCR identified one large deletion. Seven of these 11 mutations were novel (indicated with *). Two patients were found to carry mutations in the homozygous state. To confirm these findings, their parents will have to be investigated in order to exclude the presence of a large gene deletion in one of the alleles. Interestingly, the large deletion involving exons 1-3, which was previously reported in the Hungarian population, was also found in 2 unrelated patients. Two missense mutations were identified, both involving a cysteine residue, further suggesting the importance of these residues in the correct folding/processing/secretion of the neo-synthesized VWF. In those patients who still remain uncharacterized further analysis should be performed to search for intronic mutations or heterozygous large deletions responsible for aberrant splicing/post-transcriptional events. ConclusionBased on these preliminary data, HRM analysis, to our knowledge used for the first time in the molecular diagnosis of VWD3, in our hands seems to be an accurate and rapid method for mutational screening of VWF gene. However, so far, the presence of many polymorphic sites in the VWF coding region has strongly limited the use of this technique to 21 exons of the gene. Disclosures:Baronciani:Bayer Awards: Research Funding.

Clinical and molecular features in patients with atypical teratoid rhabdoid tumor or malignant rhabdoid tumor

The SMARCB1 gene status in 50 patients with atypical teratoid rhabdoid tumor and/or malignant rhabdoid tumor recruited to a German registry was prospectively analyzed with FISH and PCR. Altogether we found 40 SMARCB1 mutations in 28 patients. Two patients were positive for SMARCB1 staining at immunochemistry. Germline mutations were identified in 10 of 41 patients with CNS disease, including three large heterozygous deletions, six truncating mutations and one donor splice site mutation. No missense mutation was identified. Analysis of first degree relatives did not detect any carriers. Mutations were distributed over the SMARCB1-gene without particular clustering. No germline mutation was found in nine patients without CNS disease. Patients with germline mutation had a lower median age at diagnosis in comparison to those without detectable germline mutation (5.5 vs. 13 months, P = 0.001), a higher rate of primary multicentric CNS disease (5/10 vs. 5/36) and synchronous or metachronous mixed CNS and extracranial disease (4/10 vs. 1/36). Two year overall survival was 0% in patients with germline mutation and 48% in those without detectable germline mutation (P < 0.001). Patients with germline mutation of SMARCB1 manifest at an early age and have a very high risk for progression which has to be considered with respect to the outcome of further treatment studies.

Expanding CEP290 mutational spectrum in ciliopathies

Ciliopathies are an expanding group of rare conditions characterized by multiorgan involvement, that are caused by mutations in genes encoding for proteins of the primary cilium or its apparatus. Among these genes, CEP290 bears an intriguing allelic spectrum, being commonly mutated in Joubert syndrome and related disorders (JSRD), Meckel syndrome (MKS), Senior-Loken syndrome and isolated Leber congenital amaurosis (LCA). Although these conditions are recessively inherited, in a subset of patients only one CEP290 mutation could be detected. To assess whether genomic rearrangements involving the CEP290 gene could represent a possible mutational mechanism in these cases, exon dosage analysis on genomic DNA was performed in two groups of CEP290 heterozygous patients, including five JSRD/MKS cases and four LCA, respectively. In one JSRD patient, we identified a large heterozygous deletion encompassing CEP290 C-terminus that resulted in marked reduction of mRNA expression. No copy number alterations were identified in the remaining probands. The present work expands the CEP290 genotypic spectrum to include multiexon deletions. Although this mechanism does not appear to be frequent, screening for genomic rearrangements should be considered in patients in whom a single CEP290 mutated allele was identified.

Detection by multiplex ligation-dependent probe amplification of large deletion mutations in the COL4A5 gene in female patients with Alport syndrome

Sirs, Recently, we reported on two female subjects with Xlinked Alport syndrome (XLAS) whose genetic test findings were negative by the direct sequencing method [1]. However, we could detect their large heterozygous deletion mutations with the semi-quantitative polymerase chain reaction (PCR) method, which we frequently use these days because the technique is very simple, consisting of PCR and capillary electrophoresis [1–3]. On the other hand, this method needs sophisticated equipment for capillary electrophoresis, and every exon analysis must be conducted separately. In connection with the first submission of our last article, one of the reviewers suggested we should validate the results with the multiplex ligation-dependent probe amplification (MLPA) method, because the MLPA kit for XLAS has become commercially available. Although, at that time, there were no reports on the use of MLPA for XLAS, Hertz et al. have recently reported on the usefulness of this method [4]. This prompted us to conduct the MLPA analysis according to Hertz’s recommendation, using the SALSA P191/192 Alport MLPA assay (MRC-Holland, Amsterdam, The Netherlands) for the patients we reported on last time, and this confirmed our results. Peaks for each PCR product were obtained after capillary sequences, and the peak of every sample was determined from the relative peak area (PRA). The individual peak areas in relation to the total of all peak areas of several other parts on the X-chromosome, except for the COL4A5 and COL4A6 genes, were calculated, and the resultant values were compared to those of normal controls. We obtained values for the normal controls by calculating separately the average values for three healthy male and female subjects. A reduction in the ratio to 0 in male subjects and 0.5 in female subjects indicates a deletion in hemizygous and heterozygous form, respectively. A normal MLPA ratio is >0.7 and <1.3 [4]. We have previously detected with semi-quantitative PCR that patient 1 had an exon1-51 entire heterozygous deletion of the COL4A5 gene, patient 2 an exon 37 to 51 hemizygous deletion, and the mother of patient 2 a heterozygous deletion [1]. The MLPA results were exactly the same as those for semi-quantitative PCR (The schema is shown in Fig. 1). Moreover, the deletion in patient 1 extended to COL4A6 exons 1 and 2. XLAS associated with diffuse leiomyomas [DL-AS; online Mendelian inheritance in man (OMIM) catalogue no. 308940] has been reported previously in more than 30 cases. This phenotype is caused by deletion of the COL4A5 and extends into the second intron of the COL4A6 gene, and it should be noted that defects in the COL4A6 gene alone do not appear to cause AS or leiomyomas [5, 6]. COL4A5 and COL4A6 are arranged head-to-head at Xq22.3 Pediatr Nephrol (2009) 24:1773–1774 DOI 10.1007/s00467-009-1122-0