Joyce T. Turner

A Mosaic Activating Mutation in AKT1 Associated with the Proteus Syndrome

BACKGROUND The Proteus syndrome is characterized by the overgrowth of skin, connective tissue, brain, and other tissues. It has been hypothesized that the syndrome is caused by somatic mosaicism for a mutation that is lethal in the nonmosaic state. METHODS We performed exome sequencing of DNA from biopsy samples obtained from patients with the Proteus syndrome and compared the resultant DNA sequences with those of unaffected tissues obtained from the same patients. We confirmed and extended an observed association, using a custom restriction-enzyme assay to analyze the DNA in 158 samples from 29 patients with the Proteus syndrome. We then assayed activation of the AKT protein in affected tissues, using phosphorylation-specific antibodies on Western blots. RESULTS Of 29 patients with the Proteus syndrome, 26 had a somatic activating mutation (c.49G→A, p.Glu17Lys) in the oncogene AKT1, encoding the AKT1 kinase, an enzyme known to mediate processes such as cell proliferation and apoptosis. Tissues and cell lines from patients with the Proteus syndrome harbored admixtures of mutant alleles that ranged from 1% to approximately 50%. Mutant cell lines showed greater AKT phosphorylation than did control cell lines. A pair of single-cell clones that were established from the same starting culture and differed with respect to their mutation status had different levels of AKT phosphorylation. CONCLUSIONS The Proteus syndrome is caused by a somatic activating mutation in AKT1, proving the hypothesis of somatic mosaicism and implicating activation of the PI3K-AKT pathway in the characteristic clinical findings of overgrowth and tumor susceptibility in this disorder. (Funded by the Intramural Research Program of the National Human Genome Research Institute.).

Reassessment of the Proteus syndrome literature: Application of diagnostic criteria to published cases†

The medical care of patients affected by rare disorders depends heavily on experiences garnered from prior cases, including those patients evaluated by the treating physician and those published in the medical literature. The utility of published cases is wholly dependent upon accurate diagnosis of those patients. In our experience, the rate of misdiagnosis in Proteus syndrome (PS) is high. Diagnostic criteria have been published, but these criteria have not been applied consistently and were published after many case reports appeared in the literature. We reviewed 205 cases of individuals reported to have PS in the literature and three of us independently applied the diagnostic criteria to these case reports. Our initial diagnostic congruence was 97.1% (199/205); the discrepancies in six cases were easily resolved. Only 97 (47.3%) of reported cases met the diagnostic criteria for PS; 80 cases (39%) clearly did not meet the criteria; and although 28 cases (13.7%) had features suggestive of PS, there were insufficient clinical data to make a diagnosis. Reported cases that met the PS criteria had a higher incidence of premature death, and other complications (scoliosis, megaspondyly, central nervous system abnormalities, tumors, otolaryngologic complications, pulmonary cystic malformations, dental and ophthalmogic complications) compared to those in the non‐Proteus group. The cases that met the criteria were more often male, which has implications for hypotheses regarding the etiology and pathophysiology of PS. We also studied the attributes that led authors to conclude the reported patients had PS when we concluded they did not. We found that two of the diagnostic criteria (disproportionate overgrowth and connective tissue nevi) were often misinterpreted. In PS, the abnormal growth is asymmetric, distorting, relentless, and occurred at a faster rate compared to the rest of the body. Furthermore, PS was associated with irregular and disorganized bone, including hyperostoses, hyperproliferation of osteoid with variable calcification, calcified connective tissue, and elongation of long bones with abnormal thinning. In contrast, non‐Proteus cases displayed overgrowth that was asymmetric but grew at a rate similar to the growth found in unaffected areas of the body. Also, the overgrowth in non‐Proteus cases was associated with normal or enlarged bones together with ballooning of the overlying soft tissues. Taken together, these data show that (1) PS diagnostic criteria sort individuals with asymmetric overgrowth into distinct groups; (2) individuals with PS were more likely to have serious complications; (3) PS affects more males than females; and 4) the published diagnostic criteria are useful for clinical care and research. This article contains supplementary material, which may be viewed at the American Journal of Medical Genetics website at http://www.interscience.wiley.com/jpages/0148‐7299/suppmat/index.html. Published 2004 Wiley‐Liss, Inc.

Newly delineated syndrome of congenital lipomatous overgrowth, vascular malformations, and epidermal nevi (CLOVE syndrome) in seven patients†

We present a series of seven patients who were previously diagnosed with Proteus syndrome, but who do not meet published diagnostic criteria for this disorder and whose natural history is distinct from that of Proteus syndrome. This newly recognized phenotype comprises progressive, complex, and mixed truncal vascular malformations, dysregulated adipose tissue, varying degrees of scoliosis, and enlarged bony structures without progressive bony overgrowth. We have named this condition congenital lipomatous overgrowth, vascular malformations, and epidermal nevi (CLOVE syndrome) on a heuristic basis. In contrast to the bony distortion so characteristic of Proteus syndrome, distortion in CLOVE syndrome occurs only following major or radical surgery. Here, we contrast differences and similarities of CLOVE syndrome to Proteus syndrome.

Molecular Analysis Expands the Spectrum of Phenotypes Associated with GLI3 Mutations

A range of phenotypes including Greig cephalopolysyndactyly and Pallister‐Hall syndromes (GCPS, PHS) are caused by pathogenic mutation of the GLI3 gene. To characterize the clinical variability of GLI3 mutations, we present a subset of a cohort of 174 probands referred for GLI3 analysis. Eighty‐one probands with typical GCPS or PHS were previously reported, and we report the remaining 93 probands here. This includes 19 probands (12 mutations) who fulfilled clinical criteria for GCPS or PHS, 48 probands (16 mutations) with features of GCPS or PHS but who did not meet the clinical criteria (sub‐GCPS and sub‐PHS), 21 probands (6 mutations) with features of PHS or GCPS and oral‐facial‐digital syndrome, and 5 probands (1 mutation) with nonsyndromic polydactyly. These data support previously identified genotype–phenotype correlations and demonstrate a more variable degree of severity than previously recognized. The finding of GLI3 mutations in patients with features of oral–facial–digital syndrome supports the observation that GLI3 interacts with cilia. We conclude that the phenotypic spectrum of GLI3 mutations is broader than that encompassed by the clinical diagnostic criteria, but the genotype–phenotype correlation persists. Individuals with features of either GCPS or PHS should be screened for mutations in GLI3 even if they do not fulfill clinical criteria. Hum Mutat 31:1142–1154, 2010.

Proteus syndrome: Misdiagnosis with PTEN mutations

In this editorial, we briefly (1) define Proteus syndrome; (2) analyze reports of PTEN mutations claimed to have ‘‘Proteus syndrome’’ or a ‘‘Proteus-like syndrome’’; (3) demonstrate the high frequency of misdiagnosis of Proteus syndrome by clinicians less familiar with the disorder; and (4) discuss two series of patients who do meet the diagnostic criteria for Proteus syndrome among whom none have been found to have PTENmutations. Proteus syndrome is a highly variable disorder with strikingly relentless asymmetric and disproportionate overgrowth of body parts, cerebriform connective tissue nevi, epidermal nevi, disregulated adipose tissue, and vascular malformations [Cohen and Hayden, 1979; Wiedemann et al., 1983; Cohen et al., 2002]. The cause or causes are unknown. The currentworking hypothesis is that Proteus syndrome arises from a postzygotic mutation based on (1) mosaic distribution of lesions, (2) sporadic occurrence, (3) exclusively unaffected offspring born to affected individuals, and (4) discordant identical twins [Happle, 1987;Cohen, 1993;Biesecker et al., 1998, 1999; Cohen et al., 2002]. No other model has been proposed that would account for these observations. For reasons to be explained, we are of critical of (1) reported cases said to have Proteus syndrome with PTEN germ line mutations [Zhou et al., 2001; Smith et al., 2002] and (2) reported PTEN mutations, using the unhelpful and confounding clinical term ‘‘Proteuslike syndrome’’ [Zhou et al., 2000, 2001]. We do not dispute the PTEN mutations found per se, but the clinical diagnosisofProteussyndrome.Zhouetal. [2000]studied five patients said to have Proteus syndrome and one patient with a ‘‘Proteus-like syndrome.’’ No mutations were found in the five ‘‘Proteus syndrome’’ patients. A germline mutation was found and tissue samples showed loss of heterozygosity in the ‘‘Proteus-like’’ patient. Clinical features included ‘‘marked hypertrophyof the right lower extremity in girthand length, pink verrucoid epidermal naevi . . .with . . .plaques on the right side of his body, and macrocephaly.’’ He also had lipomas and ‘‘invasive arteriovenous malformations involving the muscles and bones of the entire right lower extremity, pelvis, lower abdomen, and buttocks, as well as diffuse verrucoid epidermal naevi over his hands, legs, and face.’’ He developed ‘‘progressive heart failure’’ and also had ‘‘hypothyroidism.’’ Zhou et al. [2000] specifically stated that their patient did not have Proteus syndrome but labeled him as having a ‘‘Proteuslike syndrome.’’ Zhou et al. [2001] then studied 14 patients from several academic medical centers in the United States and Europe. PTEN germline mutations were found in two of nine Proteus syndrome patients said to have met the diagnostic criteria for PS. Three of the five patients with a ‘‘Proteus-like syndrome’’ were also found to have PTEN germline mutations. Zhou et al. [2001] provide insufficient data about their two cases of ‘‘Proteus syndrome’’ to be validated. In their table of listed findings, one patient (their PS2) had insufficient findings for the diagnosis of Proteus syndrome [Biesecker et al., 2001]. More comprehensive clinical data were not available because of issues of informed consent [Biesecker et al., 2001; Eng et al., 2001]. The term ‘‘Proteus-like syndrome’’ applied to 13 patients by Zhou et al. [2001] has already been discussed above. Smith et al. [2002] reported a PTEN mutation in a patient said to have ‘‘classical Proteus syndrome.’’ Findings included an extensive epidermal nevus involving the left arm, hand, chest, and flank; widespread capillary malformations of the chest, abdomen, and right leg; andevidence of disproportionate overgrowthof the right leg. Many findings reported by Smith et al. [2002] indicate that their patient does not have Proteus syndrome. The following features of their propositus have never been seen in Proteus syndrome patients by us; lipoblastomatosis (not the same as lipomas which their patient also had); multiple sessile polypoid lesions of the jejunum and colon (characteristic of the PTEN hamartomatumor syndrome); and apparently a true hemangioma in addition to vascular malformations. We are unimpressed by the degree of ‘‘disproportionate overgrowth’’ shown in their patient. The combination of polyposis, lipomas, vascular malformations, and hemangiomas has been reported in the PTEN hamartoma-tumor syndrome [Cohen et al., 2002]. We have evaluated over 200 patients claimed to have Proteus syndrome from the literature or from referrals for consultation. This analysiswill be published in detail elsewhere [Turner et al., 2003]. Briefly, by applying the published diagnostic criteria for Proteus syndrome [Biesecker et al., 1999], only about half of these patients actually had Proteus syndrome. By using independent *Correspondence to: Dr. M. Michael Cohen, Jr., Dalhousie University, Halifax, Nova Scotia, Canada B3H 3J5. E-mail: r.maclean@dal.ca

Clinical and molecular delineation of the Greig cephalopolysyndactyly contiguous gene deletion syndrome and its distinction from acrocallosal syndrome

Greig cephalopolysyndactyly syndrome (GCPS) is caused by haploinsufficiency of GLI3 on 7p13. Features of GCPS include polydactyly, macrocephaly, and hypertelorism, and may be associated with cognitive deficits and abnormalities of the corpus callosum. GLI3 mutations in GCPS patients include point, frameshift, translocation, and gross deletion mutations. FISH and STRP analyses were applied to 34 patients with characteristics of GCPS. Deletions were identified in 11 patients and the extent of their deletion was determined. Nine patients with deletions had mental retardation (MR) or developmental delay (DD) and were classified as severe GCPS. These severe GCPS patients have manifestations that overlap with the acrocallosal syndrome (ACLS). The deletion breakpoints were analyzed in six patients whose deletions ranged in size from 151 kb to 10.6 Mb. Junction fragments were found to be distinct with no common sequences flanking the breakpoints. We conclude that patients with GCPS caused by large deletions that include GLI3 are likely to have cognitive deficits, and we hypothesize that this severe GCPS phenotype is caused by deletion of contiguous genes.

Hypothalamic Hamartomas and Seizures: Distinct Natural History of Isolated and Pallister-Hall Syndrome Cases

Summary:  Purpose: Hypothalamic hamartomas (HHs) have been associated with uncontrolled seizures, and aggressive therapy including surgery is often recommended. However, some patients, particularly those with other findings associated with Pallister‐Hall syndrome (PHS), have a more benign course.

Gonadal mosaicism in severe Pallister-Hall syndrome

Pallister–Hall syndrome (PHS, MIM #146510) is characterized by central and postaxial polydactyly, hypothalamic hamartoma (HH), bifid epiglottis, imperforate anus, renal abnormalities, and pulmonary segmentation anomalies. It is inherited in an autosomal dominant pattern. Here, we describe a family with two affected children manifesting severe PHS with mental retardation, behavioral problems, and intractable seizures. Both parents are healthy, with normal intelligence, and have no malformations on physical, laryngoscopic, and cranial MRI exam. The atypical presentation of these children and the absence of parental manifestations suggested an autosomal recessive mode of inheritance or gonadal mosaicism. Sequencing of GLI3 revealed a two nucleotide deletion in exon 15 (c.3385_3386delTT) predicting a frameshift and premature stop at codon 1129 (p.F1129X) in the children while both parents have wild type alleles. Genotyping with GLI3 intragenic markers revealed that both children inherited the abnormal allele from their mother thus supporting gonadal mosaicism as the underlying mechanism of inheritance (paternity was confirmed). This is the first reported case of gonadal mosaicism in PHS. The severe CNS manifestations of these children are reminiscent of children with non‐syndromic HH who often have progressive mental retardation with behavioral problems and intractable seizures. We conclude that the phenotypic spectrum of PHS can include severe CNS manifestations and that recurrence risks for PHS should include a proviso for gonadal mosaicism, though the frequency cannot be calculated from a single case report. Published 2003 Wiley‐Liss, Inc.

Zoom-in comparative genomic hybridisation arrays for the characterisation of variable breakpoint contiguous gene syndromes.

Contiguous gene syndromes cause disorders via haploinsufficiency for adjacent genes. Some contiguous gene syndromes (CGS) have stereotypical breakpoints, but others have variable breakpoints. In CGS that have variable breakpoints, the extent of the deletions may be correlated with severity. The Greig cephalopolysyndactyly contiguous gene syndrome (GCPS-CGS) is a multiple malformation syndrome caused by haploinsufficiency of GLI3 and adjacent genes. In addition, non-CGS GCPS can be caused by deletions or duplications in GLI3. Although fluorescence in situ hybridisation (FISH) can identify large deletion mutations in patients with GCPS or GCPS-CGS, it is not practical for identification of small intragenic deletions or insertions, and it is difficult to accurately characterise the extent of the large deletions using this technique. We have designed a custom comparative genomic hybridisation (CGH) array that allows identification of deletions and duplications at kilobase resolution in the vicinity of GLI3. The array averages one probe every 730 bp for a total of about 14 000 probes over 10 Mb. We have analysed 16 individuals with known or suspected deletions or duplications. In 15 of 16 individuals (14 deletions and 1 duplication), the array confirmed the prior results. In the remaining patient, the normal CGH array result was correct, and the prior assessment was a false positive quantitative polymerase chain reaction result. We conclude that high-density CGH array analysis is more sensitive than FISH analysis for detecting deletions and provides clinically useful results on the extent of the deletion. We suggest that high-density CGH array analysis should replace FISH analysis for assessment of deletions and duplications in patients with contiguous gene syndromes caused by variable deletions.

A female with complete lack of Müllerian fusion, postaxial polydactyly, and tetralogy of fallot: genetic heterogeneity of McKusick-Kaufman syndrome or a unique syndrome?

We report a 19‐year‐old, non‐Amish Caucasian female patient with primary amenorrhea caused by complete lack of Müllerian fusion with vaginal agenesis or Müllerian aplasia (MA), postaxial polydactyly (PAP), and tetralogy of Fallot. The genital tract anomaly of MA with and without renal or skeletal anomalies comprises Mayer–Rokitansky–Kuster–Hauser syndrome, which has not been reported with tetralogy of Fallot. The phenotypic triad of anomalies most closely resembled McKusick–Kaufman syndrome (MKS; OMIM 236700), a rare multiple congenital anomaly syndrome comprised of hydrometrocolpos (HMC), PAP, and congenital heart malformation that is inherited in an autosomal recessive pattern. While upper reproductive tract anomalies have not been reported with MKS, they have been reported with Bardet–Biedl syndrome (BBS), a syndrome that significantly overlaps with MKS. Both MKS and BBS can be caused by mutations in the MKKS or BBS6 gene on chromosome 20p12 and BBS is also associated with mutations in other genes (BBS1, BBS2, BBS4, and BBS7). To address this heterogenity, we sequenced the causative genes in MKS and BBS but no mutations in these five genes were identified. Fluorescence in situ hybridization (FISH) excluded large deletions of chromosome 20p12 and microsatellite marker studies confirmed biparental inheritance for all of the known BBS loci. The dual midline fusion defects of tetralogy of Fallot and MA suggests that either this patient has a unique syndrome with a distinct genetic etiology or that she has a genetically heterogeneous or variant form of MKS. Published 2004 Wiley‐Liss, Inc.