Eva Holmberg

Detection of chromosomal imbalances in children with idiopathic mental retardation by array based comparative genomic hybridisation (array-CGH)

Chromosomal aberrations are a common cause of multiple anomaly syndromes that include growth and developmental delay and dysmorphism. Novel high resolution, whole genome technologies, such as array based comparative genomic hybridisation (array-CGH), improve the detection rate of submicroscopic chromosomal abnormalities allowing re-investigation of cases where conventional cytogenetic techniques, Spectral karyotyping (SKY), and FISH failed to detect abnormalities. We performed a high resolution genome-wide screening for submicroscopic chromosomal rearrangements using array-CGH on 41 children with idiopathic mental retardation (MR) and dysmorphic features. The commercially available microarray from Spectral Genomics contained 2600 BAC clones spaced at approximately 1 Mb intervals across the genome. Standard chromosome analysis (>450 bands per haploid genome) revealed no chromosomal rearrangements. In addition, multi-subtelomeric FISH screening in 30 cases and SKY in 11 patients did not detect any abnormality. Using array-CGH we detected chromosomal imbalances in four patients (9.8%) ranging in size from 2 to 14 Mb. Large scale copy number variations were frequently observed. Array-CGH has become an important tool for the detection of chromosome aberrations and has the potential to identify genes involved in developmental delay and dysmorphism. Moreover, the detection of genomic imbalances of clinical significance will increase knowledge of the human genome by performing genotype-phenotype correlation.

Noonan and cardio-facio-cutaneous syndromes: two clinically and genetically overlapping disorders

Background: Noonan syndrome (NS) and cardio-facio-cutaneous syndrome (CFC) are related disorders associated with disrupted RAS/RAF/MEK/ERK signalling. NS, characterised by facial dysmorphism, congenital heart defects and short stature, is caused by mutations in the genes PTPN11, SOS1, KRAS and RAF1. CFC is distinguished from NS by the presence of ectodermal abnormalities and more severe mental retardation in addition to the NS phenotype. The genetic aetiology of CFC was recently assigned to four genes: BRAF, KRAS, MEK1 and MEK2. Methods: A comprehensive mutation analysis of BRAF, KRAS, MEK1, MEK2 and SOS1 in 31 unrelated patients without mutations in PTPN11 is presented. Results: Mutations were identified in seven patients with CFC (two in BRAF, one in KRAS, one in MEK1, two in MEK2 and one in SOS1). Two mutations were novel: MEK1 E203Q and MEK2 F57L. The SOS1 E433K mutation, identified in a patient diagnosed with CFC, has previously been reported in patients with NS. In one patient with NS, we also identified a mutation, BRAF K499E, that has previously been reported in patients with CFC. We thus suggest involvement of BRAF in the pathogenesis of NS also. Conclusions: Taken together, our results indicate that the molecular and clinical overlap between CFC and NS is more complex than previously suggested and that the syndromes might even represent allelic disorders. Furthermore, we suggest that the diagnosis should be refined to, for example, NS–PTPN11-associated or CFC–BRAF-associated syndromes after the genetic defect has been established, as this may affect the prognosis and treatment of the patients.

Detailed characterization of 12 supernumerary ring chromosomes using micro-FISH and search for uniparental disomy.

Twelve patients with varying degrees of mosaicism for a supernumerary ring chromosome were studied. The ring chromosomes were characterized using microdissection in combination with degenerate nucleotide-primed polymerase chain reaction (PCR) and reverse painting (micro-FISH). This method made it possible to determine the chromosomal origin of the ring chromosomes in detail, and thus to compare the phenotypes of similar cases. Eleven of the marker chromosomes were derived from the most proximal part of 1p, 3p, 3q, 5p, 7q, 8p, 8q, 9p, 10p and 20p. One marker chromosome had a complex origin, including the proximal and the most distal part of 20q. Eight of the families were also investigated for uniparental disomy (UPD) using microsatellite analysis. One case with maternal UPD 9 was found in a child with a ring chromosome derived from chromosome 9, r(9)(p10p12).

Diagnosis of familial amyloidotic polyneuropathy in Sweden by RFLP analysis

Genomic DNA from 17 Swedish patients with familial amyloidotic polyneuropathy (FAP), and 50 healthy controls were tested with a cDNA transthyretin probe. In seven of the patients, FAP was not reported in either of their parents. All 50 controls showed restriction fragments of 6.6 kb and 3.2 kb after cleavage with Nsil, while the 17 FAP patients showed RFLP markers of 5.1 and 1.5 kb. These observations indicate the same methionine for valine substitution at position 30 in Swedish patients with FAP as seen in patients with FAP from Japan, Portugal and FAP‐patients of Swedish descent from USA. However, the mean onset of FAP symptoms for the 17 Swedish patients was found to be significantly later than for the patients from Japan, Portugal and USA.

Primary immunodeficiency diseases: Genomic approaches delineate heterogeneous Mendelian disorders

Background: Primary immunodeficiency diseases (PIDDs) are clinically and genetically heterogeneous disorders thus far associated with mutations in more than 300 genes. The clinical phenotypes derived from distinct genotypes can overlap. Genetic etiology can be a prognostic indicator of disease severity and can influence treatment decisions. Objective: We sought to investigate the ability of whole‐exome screening methods to detect disease‐causing variants in patients with PIDDs. Methods: Patients with PIDDs from 278 families from 22 countries were investigated by using whole‐exome sequencing. Computational copy number variant (CNV) prediction pipelines and an exome‐tiling chromosomal microarray were also applied to identify intragenic CNVs. Analytic approaches initially focused on 475 known or candidate PIDD genes but were nonexclusive and further tailored based on clinical data, family history, and immunophenotyping. Results: A likely molecular diagnosis was achieved in 110 (40%) unrelated probands. Clinical diagnosis was revised in about half (60/110) and management was directly altered in nearly a quarter (26/110) of families based on molecular findings. Twelve PIDD‐causing CNVs were detected, including 7 smaller than 30 Kb that would not have been detected with conventional diagnostic CNV arrays. Conclusion: This high‐throughput genomic approach enabled detection of disease‐related variants in unexpected genes; permitted detection of low‐grade constitutional, somatic, and revertant mosaicism; and provided evidence of a mutational burden in mixed PIDD immunophenotypes.

De novo mutation in the mitochondrial ATP synthase subunit 6 gene (T8993G) with rapid segregation resulting in Leigh syndrome in the offspring

The mutation in the mitochondrial ATP synthase subunit 6 gene (ATP6 T8993G) was identified in a male infant who died at age 15 months of Leigh syndrome. He had 94% mutated mitochondrial DNA (mtDNA) in muscle and 92% in lymphocytes. His mother was healthy but had 37% mutated mtDNA in muscle and 38% in lymphocytes. The probands brother, who was also healthy, had 44% mutated mtDNA in lymphocytes. No mutated mtDNA was detected in muscle and lymphocytes from the maternal grandmother of the proband or in lymphocytes from 15 other maternal relatives, showing that the first carrier of the ATP6 T8993G mutation in this family was the mother of the proband. This study shows that this point mutation may occur at substantial levels in a carrier of a de novo mutation and rapid segregation with high levels of mutated mtDNA causing neurodegenerative disease may occur in the second generation.

Genome-wide screening using array-CGH does not reveal microdeletions/microduplications in children with Kabuki syndrome

Kabuki syndrome (KS) is a rare multiple congenital anomaly/mental retardation syndrome. It is characterized by a distinct facial appearance, mental retardation, postnatal growth retardation, skeletal anomalies, unusual dermatoglyphics and fetal fingertip pads. It has previously been speculated that KS is caused by a microdeletion or duplication. In a recent report, an interstitial microduplication of 8p22–23.1 was presented in several cases with this disorder. We investigated 10 Caucasian patients diagnosed with KS by fluorescence in situ hybridization and microsatellite markers located on 8p22–23.1. Using the same clones that were previously reported to be duplicated on chromosome 8p, we could exclude the duplication in all our patients. In addition, we performed a genome-wide screening on this group of patients using array-based comparative genomic hybridization containing BAC clones spaced at approximately 1 Mb intervals across the genome and could not find any evidence for gene dose alterations. The characteristics of KS are variable, a fact that complicates the diagnosis of this disorder. It is possible that we will find genetic heterogeneity among Kabuki patients, and therefore it is unlikely that all patients have an interstitial 8p duplication. We conclude that the etiology of KS remains to be solved and further genetic studies are necessary to delineate its genetic cause.

DGGE screening of mutations in mismatch repair genes (hMSH2 and hMLH1) in 34 Swedish families with colorectal cancer.

Hereditary non‐polyposis colorectal cancer (HNPCC) is an autosomal dominantly inherited syndrome which confers an increased risk for colorectal cancer and endometrial cancer as well as other tumors. It is caused by germline DNA mismatch repair (MMR) gene mutations in five MMR genes, hMSH2, hMLH1, hPMS1, hPMS2 and hMSH6. Finding mutations in these high risk families means that you can offer presymptomatic carrier diagnosis and thereby identify individuals with a very high risk for cancer. These persons benefit from counseling and should be offered surveillance. We have used DGGE to screen members from 34 families for mutations in hMLHl and hMSH2. Six mutations in five families were found, five of these mutations are new. Besides, three new polymorphisms were identified. The mutations were found in two of seven Amsterdam criteria HNPCC families and in three of four families with at least one case of early onset of CRC (before 35), suggesting there are apporopriate families to be chosen for mutation screening in MMR genes.

Screening families with endometrial and colorectal cancers for germline mutations

Editor—Endometrial cancer is the most commonly diagnosed cancer of the female reproductive tract in the United States and other western countries.1 Although several genes may be altered in these cancers,2 the molecular events in the development of endometrial carcinoma remain poorly defined. Changes in simple sequence repeats in tumour DNA relative to normal DNA, referred to as microsatellite instability (MSI), are a feature of many endometrial carcinomas.3-7 MSI occurs as a result of failing DNA mismatch repair8 and is known to accompany defects in the MLH1 , MSH2 , MSH6 , MSH3 , PMS2 , and possibly PMS1 genes. Apart from MSH3 , all these genes are associated with inherited cancer susceptibility in the context of hereditary non-polyposis colorectal cancer (HNPCC).3 9-16Endometrial carcinomas are the most common extracolonic cancers in HNPCC17 and usually occur at an early age.18Women who carry HNPCC mutations have a 22-43% lifetime risk of developing endometrial cancer as compared with 3% for the general population.19-21 According to a recent report, germline mutations of the DNA mismatch repair gene MSH6 might be specifically associated with susceptibility to endometrial cancer.22 PTEN is a newly isolated tumour suppressor gene located on chromosome 10q23, a region frequently deleted in multiple types of human cancer.23-25 Inactivation of PTEN is the underlying cause of familial Cowden disease.26 Inactivating mutations in the PTEN gene are frequently found in multiple tumour types including brain, breast, prostate, endometrial, and skin carcinomas.23-25 Knockout mice for PTEN die as early embryos, while animals heterozygous for a mutant PTEN allele develop a broad spectrum of tumours.27-29 These observations have established that PTEN has multiple target organs including the endometrium. Recent reports have shown that β-catenin is a multifunctional protein involved in …

The use of intragenic polymorphisms in determination of the genomic relevance of whole-exon deletions in MLH1 and MSH2

Fig. 1. A. Sequence analysis revealed that exon 4-11 of MLH1 was deleted in family 34. B. Sequencing of genomic DNA showed that father and daughter did not share any allele in the deleted region. C. Genomic DNA sequence shows that proband in family 88 was heterozygote at a common polymorphic site, in exon 15 of MLH1 To the Editor: Inherited susceptibility appears to account for approximately 1–5% of colorectal cancer cases, with HNPCC being one of the most common manifestations of hereditary colorectal cancer. HNPCC is caused by germline mutations in one of five DNA mismatch repair genes (MMR) MSH2, MLH1, PMS1, PMS2 and MSH6 (1). To date, more than 220 different predisposing mutations in these genes have been characterized in HNPCC patients, the majority of which occur in MSH2 and MLH1 (http://www.nfdht.nl/index.htm). Genomic large deletions in MSH2 have been reported to represent a frequent cause of HNPCC (2–4). We have investigated 142 families for mutation spectra in the MLH1 and MSH2 genes with a number of mutation detection techniques, such as DGGE/CDGE, RT-PCR, IVSP and Southern blot analysis (5). Three germline mutations in three HNPCC subjects were found as deleted transcripts in MLH1 and MSH2 by RT–PCR coupled IVSP (5). Mutations identified in RNA need to be confirmed on a genomic level before they can be counseled and used in presymptomatic testing. In family 167, a mutation representing a deletion of exons 7-10 was found on RNA, and using NsiI and HindIII digested genomic DNA on a Southern blot demonstrated an extra band compared to the normal controls (not shown). This aberrant band was not detected in anonymized blood samples from 100 healthy controls, suggesting that this variant should be considered a loss of function mutation, rather than a polymorphism, and is likely to correspond to the alteration identified in RNA in this patient. In family 34, the mutation was identified in the father, and it was possible to obtain a sample from the daughter also, representing both RNA and DNA. The cDNA deletion of exons 4-11 in MLH1 co-segregated in both affected subjects, supporting the alteration as the cause of disease (Fig. 1A). In our previous study (6), one common polymorphism was found in exon 8 of MLH1. Direct sequencing of exon 8 of MLH1