Experience of a multidisciplinary task force with exome sequencing for Mendelian disorders.

BACKGROUND: In order to optimally integrate the use of high-throughput sequencing (HTS) as a tool in clinical diagnostics of likely monogenic disorders, we have created a multidisciplinary "Genome Clinic Task Force" at the University Hospitals of Geneva, which is composed of clinical and molecular geneticists, bioinformaticians, technicians, bioethicists, and a coordinator. METHODS AND RESULTS: We have implemented whole exome sequencing (WES) with subsequent targeted bioinformatics analysis of gene lists for specific disorders. Clinical cases of heterogeneous Mendelian disorders that could potentially benefit from HTS are presented and discussed during the sessions of the task force. Debate concerning the interpretation of identified variants and the content of the final report constitutes a major part of the task force's work. Furthermore, issues related to bioethics, genetic counseling, quality control, and reimbursement are also addressed. CONCLUSIONS: This multidisciplinary task force has enabled us to create a platform for regular exchanges between all involved experts in order to deal with the multiple complex issues related to HTS in clinical practice and to continuously improve the diagnostic use of HTS. In addition, this task force was instrumental to formally approve the reimbursement of HTS for molecular diagnosis of Mendelian disorders in Switzerland.

De novo reciprocal translocation t(5;6)(q13;q34) in cattle: cytogenetic and molecular characterization.

The cytogenetic analysis of a phenotypically normal bull from the Marchigiana breed revealed the presence of an abnormal karyotype due to the presence of a very long chromosome. This finding, identified in all the metaphases observed, was associated with the 2n = 60, XY karyotype, suggesting the presence of a reciprocal translocation. RBG- banding analyses identified a de novo reciprocal translocation involving BTA5 and BTA6, t(5;6)(q13;q34), while FISH analyses using cattle-specific BACs as probes enabled the confirmation and narrowed down the breakpoint regions. Array-CGH analysis also established that neither deletions nor duplications were present in the regions including the breakpoints, nor were they present elsewhere in the genome, confirming the balanced state of the translocation.

Molecular cytogenetic characterization of the first reported case of an inv dup (4p)(p15.1-pter) with a concomitant 4q35.1-qter deletion and normal parents.

Inverted duplications associated with terminal deletions are complex anomalies described in an increasing of chromosome ends. We report on the cytogenetic characterization of the first de novo inv dup del(4) with partial 4p duplication and 4q deletion in a girl with clinical signs consistent with "recombinant 4 syndrome". This abnormality was suspected by banding, but high-resolution molecular cytogenetic investigations allowed us to define the breakpoints of the rearrangement. The terminal duplicated region extending from 4p15.1 to the telomere was estimated to be 29.27 Mb, while the size of the terminal deletion was 3.114 Mb in the 4q35.1 region. Until now, 10 patients with duplicated 4p14-p15 and deleted 4q35 chromosome 4 have been described. In all cases the abnormal chromosome 4 was derived from a pericentric inversion inherited from one of the parents. In conclusion, we have identified the first case of inv dup del(4) with normal parents suggesting that, often, terminal duplications or terminal deletions mask complex rearrangements.

Molecular characterization of Xp chromosome deletion in a fertile cow.

A young cow of the Marchigiana breed (central Italy) with normal body conformation and external genitalia underwent routine cytogenetic analyses prior to its use for reproduction. After normal chromosome staining, only one X chromosome was observed with a normal diploid number (2n = 60) in all 200 studied cells. Subsequent cytogenetic analyses by using both CBA- and RBA-banding techniques evidenced that almost all the p arms of the other X chromosome was lacking. Detailed FISH-mapping analyses with BAC covering this Xp arm region demonstrated that this large chromosome region was deleted. RBA-banding showed that the deleted X was late replicating. CGH array analysis evidenced that deletion involves the Xp arm from the telomere to around 39.5 Mb, referring to the BosTau6 cattle genome assembly. This abnormality deletes about 40 Mb of the X chromosome sequence, but, despite the large number of genes deleted, none of them are programmed to escape from inactivation. This can explain the normal phenotype of the female which is actually pregnant. Finally, we evidenced, by analysis of an SNP mapped to the deleted region (SNP rs29024121), that the only normal (e.g. nondeleted) X chromosome present derives from the father. Hence, the deletion has a maternal origin.

Pretransplant HLA mistyping in diagnostic samples of acute myeloid leukemia patients due to acquired uniparental disomy.

Although acquired uniparental disomy (aUPD) has been reported in relapse acute myeloid leukemia (AML), pretransplant aUPD involving chromosome 6 is poorly documented. Such events could be of interest because loss of heterozygosity (LOH) resulting from aUPD in leukemic cells may lead to erroneous results if HLA typing for hematopoietic stem cell donor searches is performed on blood samples drawn during blastic crisis. We report here six AML patients whose HLA typing was performed on DNA extracted from peripheral blood obtained at diagnosis. We observed LOH involving the entire HLA region (three patients), HLA-A, B, C (two patients) and HLA-A only (one patient). An array-comparative genomic hybridization showed that copy number was neutral for all loci, thus revealing partial aUPD of chromosome 6p21. When HLA typing was performed on remission blood samples both haplotypes were detected. A 3-4% LOH incidence was estimated in AML patients with high blast counts. Based on DNA mixing experiments, we determined by PCR sequence-specific oligonucleotide hybridization on microbeads arrays a detection threshold for HLA-A, B, DRB1 heterozygosity in blood samples with <80% blasts. Because aUPD may be partial, any homozygous HLA result should be confirmed by a second typing performed on buccal swabs or on blood samples from the patient in remission.

Megacystis, megacolon, and malrotation: a new syndromic association?

Chronic intestinal pseudo-obstruction (CIPO) can occur as a consequence of neuropathies including diffuse Intestinal Neuronal Dysplasia (IND), a relatively rare enteric nervous system (ENS) abnormality. Although various authors reported of diffuse IND associated either with intestinal malrotation or megacystis, the co-existence of these three entities in the same patient has never been described before. The aim of this paper is to report for the first time in literature a series of patient with such association, focusing on one who carries a de novo duplication of chromosome 12, suggesting a new syndromic association (megacolon, megacystis, malrotation).

A de novo 12q13.11 microdeletion in a patient with severe mental retardation, cleft palate, and high myopia.

We report a de novo 12q13.11 deletion of 1.3 Mb in an 10-year-old dysmorphic girl with a multiple congenital anomalies/mental retardation (MCA/MR) syndrome consisting mainly of severe mental retardation, cleft palate, and high myopia. The deleted region encompasses 16 RefSeq genes. Among these, it is hypothesized that haploinsufficiency of AMIGO2 is potentially responsible for the mental retardation of this patient, and of COL2A1 for the cleft palate and high myopia.

Reciprocal translocation t(4;7)(q14;q28) in cattle: molecular characterization.

Cytogenetic analysis of a phenotypically normal young bull from the Marchigiana breed revealed the presence of an abnormal chromosome. The finding of one oversize chromosome in all metaphases, associated with a 2n = 60, XY karyotype, suggested that a reciprocal translocation had occurred. RBG-banding and FISH analyses, using specific bovine BAC probes, identified a de novo reciprocal translocation t(4;7)(q14;q28). The presence of rcp(4;7) was confirmed by FISH experiments using BTA4 and BTA7 whole chromosome probes. An array-CGH analysis (Agilent 244A) using a bovine custom design was performed to investigate if the translocation was associated with loss or gain of genetic material. The absence of a concomitant deletion or duplication at the break points allowed the balanced state of the translocation to establish. The analysis also revealed the presence of several CNVs throughout the genome. To our knowledge this is the first time the balanced condition of a cattle RCP has been ascertained using the array-CGH approach.

De novo duplication of MECP2 in a girl with mental retardation and no obvious dysmorphic features.

Loss-of-function mutations of MECP2 are responsible for Rett syndrome (RTT), an X-linked neurodevelopmental disorder affecting mainly girls. The availability of MECP2 testing has led to the identification of such mutations in girls with atypical RTT features and the recognition of milder forms. Furthermore, duplication of the entire gene has recently been described in boys with mental retardation and recurrent infections. We describe a girl with a heterozygous de novo MECP2 duplication. The patient, at the age of 19, has mental retardation with no autistic features. She is friendly but gets frequently anxious. She has neither dysmorphic features nor malformations. Her motor development was delayed with walking at 20 months. Speech is fluid with good pronunciation but is simple and repetitive. Diagnosis was made after single-strand conformation analysis (SSCA) and multiplex ligation-dependent probe amplification (MLPA) analysis of MECP2. Array comparative genomic hybridization (aCGH) analysis showed a duplication of 29 kb including MECP2 and part of IRAK1. Fluorescent in situ hybridization (FISH) has revealed that the duplicated region is inserted near the telomere of the short arm of chromosome 10. X-chromosome inactivation in leukocyte DNA was not skewed. We conclude that it is likely that this MECP2 duplication is responsible for the mental retardation in this patient. This case broadens the phenotypic spectrum of MECP2 abnormalities with consequent implication in diagnosis and genetic counselling of girls with non-syndromic mental retardation.

Duplications in addition to terminal deletions are present in a proportion of ring chromosomes: clues to the mechanisms of formation.

BACKGROUND AND METHODS: Ring chromosomes are often associated with abnormal phenotypes because of loss of genomic material at one or both ends. In some cases no deletion has been detected and the abnormal phenotype has been attributed to mitotic ring instability. We investigated 33 different ring chromosomes in patients with phenotypic abnormalities by array based comparative genomic hybridisation (CGH) and fluorescence in situ hybridisation (FISH). RESULTS: In seven cases we found not only the expected terminal deletion but also a contiguous duplication. FISH analysis in some of these cases demonstrated that the duplication was inverted. Thus these ring chromosomes derived through a classical inv dup del rearrangement consisting of a deletion and an inverted duplication. DISCUSSION: Inv dup del rearrangements have been reported for several chromosomes, but hardly ever in ring chromosomes. Our findings highlight a new mechanism for the formation of some ring chromosomes and show that inv dup del rearrangements may be stabilised not only through telomere healing and telomere capture but also through circularisation. This type of mechanism must be kept in mind when evaluating possible genotype-phenotype correlations in ring chromosomes since in these cases: (1) the deletion may be larger or smaller than first estimated based on the size of the ring, with a different impact on the phenotype; and (2) the associated duplication will in general cause further phenotypic anomalies and might confuse the genotype-phenotype correlation. Moreover, these findings explain some phenotypic peculiarities which previously were attributed to a wide phenotypic variation or hidden mosaicism related to the instability of the ring.

Cryptic deletions are a common finding in "balanced" reciprocal and complex chromosome rearrangements: a study of 59 patients.

Using array comparative genome hybridisation (CGH) 41 de novo reciprocal translocations and 18 de novo complex chromosome rearrangements (CCRs) were screened. All cases had been interpreted as "balanced" by conventional cytogenetics. In all, 27 cases of reciprocal translocations were detected in patients with an abnormal phenotype, and after array CGH analysis, 11 were found to be unbalanced. Thus 40% (11 of 27) of patients with a "chromosomal phenotype" and an apparently balanced translocation were in fact unbalanced, and 18% (5 of 27) of the reciprocal translocations were instead complex rearrangements with >3 breakpoints. Fourteen fetuses with de novo, apparently balanced translocations, all but two with normal ultrasound findings, were also analysed and all were found to be normal using array CGH. Thirteen CCRs were detected in patients with abnormal phenotypes, two in women who had experienced repeated spontaneous abortions and three in fetuses. Sixteen patients were found to have unbalanced mutations, with up to 4 deletions. These results suggest that genome-wide array CGH may be advisable in all carriers of "balanced" CCRs. The parental origin of the deletions was investigated in 5 reciprocal translocations and 11 CCRs; all were found to be paternal. Using customized platforms in seven cases of CCRs, the deletion breakpoints were narrowed down to regions of a few hundred base pairs in length. No susceptibility motifs were associated with the imbalances. These results show that the phenotypic abnormalities of apparently balanced de novo CCRs are mainly due to cryptic deletions and that spermatogenesis is more prone to generate multiple chaotic chromosome imbalances and reciprocal translocations than oogenesis.

A large analphoid invdup(3)(q22.3qter) marker chromosome characterized by array-CGH in a child with malformations, mental retardation, ambiguous genitalia and Blaschko's lines.

We report a new case of mosaic chromosome 3-derived marker chromosome, present in fibroblasts but not in lymphocytes, found in a child with malformations, mental retardation and ambiguous genitalia. Cytogenetic and molecular analysis showed that the supernumerary invdup(3)(q22.3qter) chromosome was negative at FISH with alpha satellite probe. The presence of a functional neocentromere was confirmed by immunofluorescence with antibodies to centromere proteins (CENPs). Definition of the marker breakpoints has been done through array-CGH. The skin of the patient presented dyschromic areas ordered along Blaschko's lines. The invdup(3q) marker chromosome was present only in fibroblasts from the dark skin biopsy, while lymphocytes and fibroblasts from the normal skin showed a normal male karyotype. Expression of the HPS3 gene (MIM: 606118) was more than two times higher in dark skin fibroblasts. Neocentromeres are most often observed on chromosomal arm fragments that have separated from an endogenous centromere, and therefore actually confer mitotic stability to what would have been acentric fragments. To our knowledge, this invdup(3q) analphoid marker is the largest among the several reported so far. Parental origin and possible mode of formation have been defined by DNA polymorphisms studies. The size of the duplicated marker chromosome and its frequency and tissue distribution may be relevant to the severity of the propositus' phenotype.

2q24-q31 deletion: report of a case and review of the literature.

We report a patient with a de novo interstitial deletion of the long arm of chromosome 2 involving bands 2q24.3-q31.1. The patient shows postnatal growth retardation, microcephaly, ptosis, down-slanting palpebral fissures, long eyelashes and micrognathia. Halluces are long, broad and medially deviated, while the other toes are laterally deviated and remarkably short with hypoplastic phalanges. She also showed developmental delay, seizures, lack of eye contact, stereotypic and repetitive hand movements and sleep disturbances with breath holding. Prenatal and three independent postnatal karyotypes were normal. Array-CGH analysis allowed us to identify and characterize a "de novo" 2q interstitial deletion of about 10.4Mb, involving segment between cytogenetic bands 2q24.3 and 2q31.1. The deletion was confirmed by quantitative PCR. About 30 children with 2q interstitial deletion have been reported. The deletion described here is overlapping with 15 of these cases. We have attempted to compare the clinical features of our patient with 15 overlapping cases. The emerging phenotypes include low birth weight, postnatal growth retardation, mental retardation and developmental delay, microcephaly, and peculiar facial dysmorphisms. Peculiar long and broad halluces with an increased distance between the first and the second toe are ("sandal gap" sign) present in most of the described patients. The gene content analysis of the deleted region revealed the presence of some genes that may be indicated as good candidates in generating both neurological and dysmorphic phenotype in the patient. In particular, a cluster of SCNA genes is located within the deleted region and it is known that loss of function mutations in SCNA1 gene cause a severe form of epilepsy.