Angelika J. Dawson

Disorders of sex development: Insights from targeted gene sequencing of a large international patient cohort

BackgroundDisorders of sex development (DSD) are congenital conditions in which chromosomal, gonadal, or phenotypic sex is atypical. Clinical management of DSD is often difficult and currently only 13% of patients receive an accurate clinical genetic diagnosis. To address this we have developed a massively parallel sequencing targeted DSD gene panel which allows us to sequence all 64 known diagnostic DSD genes and candidate genes simultaneously.ResultsWe analyzed DNA from the largest reported international cohort of patients with DSD (278 patients with 46,XY DSD and 48 with 46,XX DSD). Our targeted gene panel compares favorably with other sequencing platforms. We found a total of 28 diagnostic genes that are implicated in DSD, highlighting the genetic spectrum of this disorder. Sequencing revealed 93 previously unreported DSD gene variants. Overall, we identified a likely genetic diagnosis in 43% of patients with 46,XY DSD. In patients with 46,XY disorders of androgen synthesis and action the genetic diagnosis rate reached 60%. Surprisingly, little difference in diagnostic rate was observed between singletons and trios. In many cases our findings are informative as to the likely cause of the DSD, which will facilitate clinical management.ConclusionsOur massively parallel sequencing targeted DSD gene panel represents an economical means of improving the genetic diagnostic capability for patients affected by DSD. Implementation of this panel in a large cohort of patients has expanded our understanding of the underlying genetic etiology of DSD. The inclusion of research candidate genes also provides an invaluable resource for future identification of novel genes.

Cryptic chromosome rearrangements detected by subtelomere assay in patients with mental retardation and dysmorphic features

The regions near telomeres of human chromosomes are gene rich. Chromosome subtelomere rearrangements occur with a frequency of 7–10% in children with mild‐to‐moderate mental retardation (MR) and approximately 50% of cases are familial. Clinical investigation of subtelomere rearrangements is now prompted by fluorescence in situ hybridization (FISH) analysis using specific DNA probes from all relevant chromosome ends. In our study, 40 children were selected for subtelomere assay using either the Chromophore Multiprobe‐T Cytocell device or the VYSIS TelVision probes. Inclusion criteria were: developmental delay or MR; a normal 550 G‐band karyotype; FRAXA negative; and at least one other clinical criterion. Exclusion criteria included an identified genetic or environmental diagnosis. Of the 40 patients analysed, four (10%) were found to have subtelomere rearrangements. Three of 40 (7.5%) were found to have an unbalanced subtelomere rearrangement and one of 40 (2.5%) was found to have an apparently normal variant subtelomere deletion. The first of the three with an unbalanced karyotype was the result of a familial translocation, the second was a de novo finding, and the origin of the third could not be determined. The subtelomere FISH assay detected almost twice the frequency of unbalanced karyotypes as those detected by 550 G‐banding in our cytogenetics laboratory (4.7%). In addition, subtelomere screening was eight times more likely than fragile X screening in our DNA laboratory (1%) to detect genetic abnormalities in mentally handicapped individuals. Our findings support the view that screening for subtelomere rearrangements has a greater positive yield than other commonly used genetic investigations and, if cost and resources permit, should be the next diagnostic test of choice in a child with unexplained MR/dysmorphisms and a normal 550 G‐band karyotype.

Understanding the impact of 1q21.1 copy number variant

Background1q21.1 Copy Number Variant (CNV) is associated with a highly variable phenotype ranging from congenital anomalies, learning deficits/intellectual disability (ID), to a normal phenotype. Hence, the clinical significance of this CNV can be difficult to evaluate. Here we described the consequences of the 1q21.1 CNV on genome-wide gene expression and function of selected candidate genes within 1q21.1 using cell lines from clinically well described subjects.Methods and ResultsEight subjects from 3 families were included in the study: six with a 1q21.1 deletion and two with a 1q21.1 duplication. High resolution Affymetrix 2.7M array was used to refine the 1q21.1 CNV breakpoints and exclude the presence of secondary CNVs of pathogenic relevance. Whole genome expression profiling, studied in lymphoblast cell lines (LBCs) from 5 subjects, showed enrichment of genes from 1q21.1 in the top 100 genes ranked based on correlation of expression with 1q21.1 copy number. The function of two top genes from 1q21.1, CHD1L/ALC1 and PRKAB2, was studied in detail in LBCs from a deletion and a duplication carrier. CHD1L/ALC1 is an enzyme with a role in chromatin modification and DNA damage response while PRKAB2 is a member of the AMP kinase complex, which senses and maintains systemic and cellular energy balance. The protein levels for CHD1L/ALC1 and PRKAB2 were changed in concordance with their copy number in both LBCs. A defect in chromatin remodeling was documented based on impaired decatenation (chromatid untangling) checkpoint (DCC) in both LBCs. This defect, reproduced by CHD1L/ALC1 siRNA, identifies a new role of CHD1L/ALC1 in DCC. Both LBCs also showed elevated levels of micronuclei following treatment with a Topoisomerase II inhibitor suggesting increased DNA breaks. AMP kinase function, specifically in the deletion containing LBCs, was attenuated.ConclusionOur studies are unique as they show for the first time that the 1q21.1 CNV not only causes changes in the expression of its key integral genes, associated with changes at the protein level, but also results in changes in their known function, in the case of AMPK, and newly identified function such as DCC activation in the case of CHD1L/ALC1. Our results support the use of patient lymphoblasts for dissecting the functional sequelae of genes integral to CNVs in carrier cell lines, ultimately enhancing understanding of biological processes which may contribute to the clinical phenotype.

Cytogenetic aspects of the Canadian early and mid-trimester amniotic fluid trial (CEMAT).

Cytogenetic results from a large multicentre randomized controlled study of 2108 amniotic fluids obtained at 11+0–12+6 weeks (EA) and 1999 fluids at 15+0–16+6 weeks (MA) were compared. There was no statistically significant difference in the rate of chromosome abnormalities (EA =1.9 per cent; MA=1.7 per cent) or level III mosaicism (EA=0.2 per cent; MA= 0.2 per cent) between the groups. Level I and Level II mosaicism occurred more frequently in MA.

Familial transmission of a small supernumerary marker chromosome 8 identified by FISH: An update

A father and his 2 daughters were previously determined to carry a small, supernumerary marker chromosome [Chudley et al., 1983]. The origin of this marker could not be determined by standard cytogenetic techniques. In this study, fluorescence in situ hybridization (FISH) studies identified the marker chromosome as a pericentric derivative of chromosome 8. The father has low grade mosaicism for this marker and is phenotypically normal. Both daughters are non-mosaic and show developmental delays and somewhat differing clinical findings. The phenotypes of the 2 sisters are compared with those previously reported for supernumerary der(8) patients. This is the first report of familial transmission of a supernumerary der(8) marker chromosome.

Proximal interstitial 6q deletion: A recognizable syndrome

We report on an 8-year-old boy with a proximal interstitial deletion of the long arm of chromosome 6 with breakpoints q13 to q14.2. He has a characteristic facial appearance that is seen in several of the previously described cases. Details of his clinical course are reviewed and compared with the nine previous reported cases of the proximal deletion 6q syndrome.

The evaluation of 15q proximal duplications by FISH.

Six patients from the clinical cytogenetics laboratory identified as having the normal variant dup(15)(q12) were further evaluated using fluorescence in situ hybridization (FISH). The purpose of this study was to ascertain whether any of the Prader–Willi/Angelman Chromosome Region (PWCR and ANCR, respectively) loci were duplicated in these patients. The results indicated that the patients could be categorized into two groups. The first group showed no duplication of the PWCR/ANCR loci and appeared to belong to the dup(15)(q12) class which is phenotypically silent and is therefore called the normal variant. The second group also showed no duplication of the PWCR/ANCR loci, but had a large alpha satellite variant with D15Z. It is hypothesized that these patients do not have a duplication of 15q12 but rather a centromeric variant which mimics the dup(15)(q12). This study confirms the importance of evaluating apparent variations in the proximal region of chromosome 15 with FISH.

Severe hemihypotrophy in a female infant with mosaic Turner syndrome: a variant of Russell–silver syndrome?

Russell–Silver syndrome is a genetically heterogeneous condition. For most affected individuals, it represents a phenotype rather than a specific disorder. Although chromosomal anomalies, imprinting disorder, maternal uniparental disomy 7 as well as familial autosomal dominant and X-linked forms have been reported, the diagnosis remains determined on clinical grounds. Russell–Silver syndrome is characterized by asymmetric intrauterine growth retardation, postnatal failure to thrive, distinct facial features, limb asymmetry, excessive sweating and minor skin lesions. We report here a female infant who had a karyotype of 45,X on prenatal amniocytes. After delivery she was noted to have features not explainable on the basis of Turner syndrome. Her phenotype actually was quite consistent with Russell–Silver syndrome. She had a triangular face with prominent forehead, large eyes, a thin nose, malar hypoplasia, thin upper lip with down-turned corner of the mouth and a pointed chin. Marked body asymmetry was evident at birth, with the left side significantly smaller than the right side. She has a diphalangeal left fifth finger. Skin fibroblast culture and analysis showed a karyotype of 45,X on the right side and 45,X/46,XX on the left side. The case is another illustration of the genetic heterogeneity of Russell–Silver phenotype.

Interstitial 4p deletion in a child with an Angelman syndrome-like phenotype

To the Editor: We present a child with a de no6o deletion of chromosome 4 involving p15.2p16.1. The phenotype is reminiscent of Angelman syndrome (AS). We recommend that other investigations are indicated in cases where AS is suspected but molecular studies of chromosome 15 are normal. Deletions of the short arm of chromosome 4 lead to specific phenotypes. Wolf–Hirschorn syndrome and Pitt–Rogers–Danks syndrome, which are distal 4p deletion syndromes, are characterized by growth retardation, developmental delay, and dysmorphic features (1–3). A preliminary phenotypic map of chromosome 4p16 has been developed (4). Patients with interstitial deletions of 4p also share a common phenotype (5). The propositus, SB, was referred for evaluation of motor delay and minor dysmorphic features. The family, pregnancy, birth, and past medical histories were unremarkable. He could not stand unsupported at 3 years of age. He was described as always being happy and affectionate. On examination he laughed throughout. He had no intelligible words and drooled excessively. His movements were unsteady. Height was at the 95th percentile, weight was at the 50th percentile and head circumference was at the 10th percentile. He had blond hair and fair skin. He had brachycephaly, a wide mouth, widely spaced teeth, and midfacial hypoplasia. His tone was normal with brisk reflexes (Fig. 1a,b). Given his presentation we suspected AS. CT scan of the brain and metabolic studies were normal. High resolution cytogenetic analysis (6), on the patient, revealed an abnormal karyotype with an interstitial deletion of the short arm of chromosome 4 with breakpoints at 4p15.2 and 4p16.1. The parental karyotypes were normal. The resulting karyotype was 46,XY,del(4)(p15.2p16.1) de no6o (Fig. 1c). Southern blot analysis was performed on the patient using XbaI/NotI restriction enzyme digest and the SNRPN probe (7). This revealed biparental chromosome 15 contributions based on methylation status (data not shown). Several patients with interstitial deletions of chromosome 4p have been described. White et al. (5) described 7 patients with deletions of 4p14 to 4p16, and at least 11 other cases have been published involving deletions of 4p12 to 4p15 (see Table 1) (8–18). Our patient has some similarities to these patients, but he did not share many of the features common to these individuals (Table 1). The minimal deleted segment proposed for the proximal interstitial 4p syndrome includes (4)(p15.2p15.33) (17), which is deleted in our patient. AS has been extensively reviewed (19–22), and a consensus statement on clinical features has been published (23). Features present in all cases include developmental delay, speech impairment, a movement or balance disorder and behavioral uniqueness (including frequent, inappropriate laughing or a happy demeanor). Approximately 80% of patients who meet the diagnostic criteria for AS will have either a cytogenetic molecular deletion or uniparental inheritance of chromosome 15. Of the remaining 20%, some will have identified UBE3A mutations (24). Of the patients with an UBE3A mutation, half are de no6o and half are maternally inherited. Other cases have been reported in which a patient with a phenotype reminiscent of AS has been found to have a different genetic defect. Mickelson et al. (25) report a patient with features of AS with a deletion of chromosome 17q. Arn et al. (26) describe a patient, previously diagnosed with AS, who was found to have methlyenetetrahydrofolate reductase deficiency. We have reviewed a child with an interstitial deletion of chromosome 4p with a unique phenotype. These differences may be explained either by different breakpoints, by parent of origin or imprinting effects, or by the effects of other genetic and environmental factors. Our findings support the performance of high resolution chromosome analysis, to exclude or identify cytogenetic abnor-

Vertical transmission of the Ohdo blepharophimosis syndrome.

Ohdo blepharophimosis syndrome (OBS) is a multiple congenital anomalies-mental retardation syndrome composed of blepharophimosis, ptosis, dental hypoplasia, partial deafness, and mental retardation. Previously reported cases of OBS have been sporadic except for the report by Ohdo et al. [1986, J Med Genet 23:242-244] that described two affected sisters and a first cousin favoring autosomal recessive inheritance. The original report by Ohdo et al. [1986] may reflect nonpenetrance of an autosomal dominantly inherited disorder or genetic heterogeneity of OBS. We report on a child and the mother who have blepharophimosis, ptosis, dental anomalies, mild hearing loss, and mental retardation. Chromosome analysis in both showed a balanced paracentric inversion of the long arm of chromosome 9, which was also present in two phenotypically normal sibs of the mother. This is the first report of vertical transmission of OBS suggestive of autosomal dominant inheritance. X-linked dominant and mitochondrial inheritance are other possible modes of inheritance.