Cornelia de Lange syndrome: Extending the physical and psychological phenotype

Authors


  • Proceedings from the 3rd Cornelia de Lange World Conference 2009.

  • How to Cite this Article: Oliver C, Bedeschi MF, Blagowidow N, Carrico CS, Cereda A, FitzPatrick DR, Gervasini C, Griffith GM, Kline AD, Marchisio P, Moss J, Ramos FJ, Selicorni A, Tunnicliffe P, Wierzba J, Hennekam RCM. 2010. Cornelia de Lange syndrome: Extending the physical and psychological phenotype. Am J Med Genet Part A 152A:1127–1135.

INTRODUCTION

The third Cornelia de Lange Syndrome World Scientific Conference was held in July 2009 in Brighton, United Kingdom. The Scientific Conference preceded a conference for professionals from health, education and other backgrounds who work with children and adults with Cornelia de Lange syndrome (CdLS) and a 2-day family conference. Delegates and families from over 20 countries attended the conferences.

The goal of the conference was to provide a forum for the exchange of research results, information on best clinical practice, and perspectives on support relevant to the wellbeing of children and adults with CdLS. The purpose of this proceeding is to provide a summary of the scientific conference to those researchers and clinicians who were unable to attend.

SUMMARY OF PRESENTATIONS

The scientific conference included presentations on molecular genetics, genotype–phenotype correlations, sensory abnormalities, physical health, aging, the social and behavioral phenotype of CdLS, and family perspectives on care and management for persons with CdLS. The diversity of research presentations is indicative of the breadth of activity and perspectives in a growing research agenda. CdLS is a complex developmental disorder with the cause identified in approximately 55–60% of those clinically diagnosed as arising from mutations of the NIPBL, SMC1A, and SMC3 genes [Krantz et al., 2004; Tonkin et al., 2004; Bhuiyan et al., 2006; Musio et al., 2006; Deardorff et al., 2007; Kline et al., 2007a]. There is evidence that NIBL mutations are associated with a more severe phenotype (Abstract 2) but there is variability in the phenotypes for each gene mutation (Abstract 15). Mutations of the PDS5A and CTCF genes have been investigated as possible causes of CdLS in those who do not have NIPBL, SMC1A, and SMC3 genes, with negative results (Abstracts 11 and 12).

Impaired growth, craniofacial and upper limb abnormalities are common in CdLS and numerous physical health problems are reported with a high prevalence of gastrointestinal disorders and feeding disorders. In a clinical cohort of 100 patients with CdLS postnatal growth was below the third centile for 84% of patients and gastroesophageal reflux (GER) was present in 73% (Abstract 14). Abdominal ultrasound has been found to be a useful adjunct to clinical examination for the purpose of diagnosis of renal, urinary, and uterine anomalies (Abstract 3). Kidney malformations are evident in approximately 30% of patients and cardiac malformations (primarily pulmonary stenosis) occur in 35% (Abstracts 13 and 14). A broad array of feeding problems is reported (Abstracts 1 and 10), including a lack of sucking reflex in neonates (56%). Audiological assessment reveals abnormal hearing in 82% of patients with conductive hearing loss being the most commonly reported problem (Abstract 6). A second study revealed 45% of patients to have either sensorineural or conductive hearing loss (Abstract 7) and that these disorders were related to the size of the NIPBL mutation. Additionally, this study reports myopia and ptosis to be associated with truncating NIPBL mutations.

Profound to severe intellectual disability is normally evident in CdLS [Oliver et al., 2008] with autism spectrum disorders reported in approximately 50–67% of children and adults [Berney et al., 1999; Bhuiyan et al., 2006; Basile et al., 2007; Moss et al., 2008] alongside impaired expressive communication, compulsive behaviors, and social anxiety [Hyman et al., 2002; Richards et al., 2009; Moss et al., 2009; Wulffaert et al., 2009]. In comparison with other genetic syndromes a lack of sociability combined with autism spectrum-like characteristics are evident even when degree of intellectual disability is controlled for (Abstract 8). Unusually high levels of self-injurious behavior with comparatively low levels of aggression are reported [Oliver et al., 2009] with increasing evidence of a relationship between self-injury and GER (Abstracts 9 and 14).

Three issues are likely to feature prominently in future research. First, genotype–phenotype correlations and the differentiation of a mild from classic phenotype is important for prognosis and understanding the effects of the different gene mutations (Abstract 5). Second, reports of significant age-related changes in the physical phenotype allude to a potential role for abnormalities in DNA repair pathways [Kline et al., 2007b] (Abstract 4). The changes with age in the physical phenotype, behavior, and neuropsychological functioning warrant examination in cross-sectional and longitudinal studies. Third, the complexity of the task facing families and professionals when trying to support children and adults with CdLS is clearly evident given the diverse and multi-system effects of the syndrome (Abstract 18). Coordination of timely service delivery is an important but infrequently explored issue.

SPEAKERS' ABSTRACTS

Perinatal and Neonatal Data in 43 Polish Patients

Jolanta Wierzba. We studied the perinatal and neonatal data in 43 live born classically affected CdLS patients recruited through the CdLS Association, Poland. Fourteen newborns (32.6%) were born prematurely, and 26 individuals (60.5%) were small for gestational age; 7 of these were born prematurely. Frequent congenital malformations were limb defects (n = 28; 65.1%) of whom 7 (16.3%) had severe upper limb reduction defects; heart malformations (n = 26; 60.5%); renal tract malformations (n = 9; 20.9%); and cleft palate (n = 11; 25.6%). Cryptorchidism was observed in all males but one. All individuals demonstrated abnormal muscular tone. Trembling and convulsions were observed incidentally. Feeding problems were frequent (n = 41; 95.3%). Twenty-four neonates (55.8%) demonstrated initially a total lack of sucking reflex. Surgical interventions in the neonate period were needed in three newborns because of a congenital heart defect with esophageal and duodenal atresia, inguinal hernia incarceration, and intestine perforation.

The data from the Polish cohort are consistent with earlier reports from both American and European studies. The multiple organs that can be affected in CdLS newborns, and the wide variability indicates the need for multidisciplinary diagnostics and intervention in the neonatal period.

Correlation of the Neonatal Phenotype and Genotype

A. Cereda, L. Colombo, A. Passarini, S. Russo, P. Castronovo, D. Milani, A. De Paoli, F Mosca, L. Memo, and A. Selicorni. CdLS is usually diagnosed at birth or during the first months of life. This period can be particularly stressful for parents, both because of the frequent medical problems and because families have to cope with the diagnosis. We focused attention on neonatal features and feeding problems of a large group of Italian CdLS patients. All the patients were studied molecularly for NIPBL and SMC1 mutations. As the number of SMC1-positive patients was small clinical data were only compared between NIPBL-positive and -negative patients.

We collected data on the neonatal period in 101 CdLS patients (57 men). In 23%, clinical diagnosis was established at birth; 75% of them had a NIPBL mutation. In 52% of patients the clinical diagnosis was established later on but within the first years of life; an NIPBL mutation was present in 63%. In patients diagnosed after the first year (25%) a NIPBL mutation was found in 29%. Prenatal growth retardation was evident in 58% (62% with a NIPBL mutation) and absent in 42% (25% with a NIPBL mutation). Mean gestational duration was 38 weeks, mean birth weight was 2,440 g in men and 2,310 g in women, and mean length at birth was 45.5 cm in men and 44.0 cm in women. Mean head circumference at birth was 31.0 cm in men and 30.2 cm in women. Data for NIPBL-positive and -negative patients were for birth weight 2,180 and 2,570 g, for length 44 and 46 cm, and for head circumference 30 and 31 cm, respectively. Some form of neonatal resuscitation was needed in 19.5% of patients (53% NIPBL-positive; if no resuscitation needed 39% was NIPBL-positive). Feeding problems were present in 71% of patients and 43% needed additional nutritional support (56% NIPBL-positive; without feeding problems 36% was NIPBL-positive).

These data confirmed that CdLS newborns with an NIPBL mutation show a more severe phenotype: prenatal growth is more reduced, birth weight and length are lower, neonatal resuscitation was more frequently needed, and feeding problems and assisted nutrition were more common. In accordance with this, the more severe neonatal phenotype in NIPBL-positive cases led to an earlier diagnosis, usually before 1 year of age.

2D and 3D Abdominal Ultrasound in Adolescents and Adults

Natalie Blagowidow, J. Camak, and K. Teagarden. Individuals with CdLS are at increased risk for a variety of medical conditions, including renal anomalies, urinary reflux, and uterine anomalies. In addition, we have observed cholelithiasis and prostate enlargement in adult patients with CdLS. Communication of symptoms is difficult for those with more significant developmental delay. Clinical examination can also be challenging. In particular, female patients may not cooperate with a gynecologic examination, resulting in few or no examinations. We elected to evaluate the utility of abdominal ultrasound in adolescent and adult CdLS patients.

We performed abdominal ultrasound in 18 consecutive individuals attending the multidisciplinary aging CdLS clinic. The physician interpreting the studies was present for all examinations. In 14 patients both 2D and 3D imaging was performed. Ten patients were studied in only the supine position, one only seated, and seven both seated and supine. Significant findings were small kidneys (n = 4), and a renal cyst, a bicornuate uterus, a fatty liver, and a calcification within the prostate (one each). Satisfactory imaging was achieved for the liver in 94%, gallbladder in 71%, spleen in 61%, kidneys in 56%, uterus in 100%, ovaries in 57%, and prostate in 50%. Images were suboptimal owing to patient inability to cooperate, and because of obesity. The 3D modality provided similar imaging as the 2D, with helpful imaging of abnormal findings. The seated position was effective for upper abdominal imaging, but the supine position provided more satisfactory images in three patients. We concluded that eight significant abnormalities were identified in 18 patients. We were able to examine several women in whom a gynecologic examination had not been tolerated. Abdominal ultrasound is a useful diagnostic tool, and can serve as an adjunct to clinical examination.

Premature Aging

Antonie D. Kline. CdLS is a variable syndrome that can present with a broad spectrum of involvement. Significant progress has been made in recent years with the clinical and molecular delineation of CdLS, contributing to new insights with respect to clinical findings, and allowing observations about natural history. Some evidence for premature aging has been found, and mechanistically this is likely to be linked to the abnormal function of cohesin owing to the gene mutations causing CdLS.

We evaluated 71 adolescent and adult patients in a multidisciplinary aging clinic for CdLS, with patients recruited through the CdLS Foundation. Medical histories and physical examinations were performed on each patient, with specialized evaluations given by the subspecialists.

The age range was 13–50 years with an equal sex ratio. There was a specific pattern of aging to the facies, with lengthening of the face, slight coarsening of features, and squaring of the chin. Facial asymmetry was also noted. Many individuals had a physical appearance older than their chronologic years. In the cohort, 5% developed Type II diabetes, 4% developed hypertension, and there was no evidence for myocardial infarctions. Clinically insignificant small pericardial effusion was seen echographically in 5 of 7 investigated patients. GER was noted in over 90% of patients, and 14% developed a Barrett esophagus, some as early as 17 years. Two percent of the patients have had cholecystitis requiring cholecystectomy. Ninety percent of a cohort of 20 ambulatory patients was found to have some degree of decreased bone density with age and 75% to have low vitamin D. Three patients had an enlarged prostate, one of which was removed by 41 years of age. No patient developed any form of cancer. Behavioral issues were prevalent, including self-injury, aggression, and anxiety, and could be debilitating. Sleep disturbance occurred in 80%.

One biologic pathway that has been linked to premature aging is that of DNA repair. Cohesin is recruited to sites of DNA damage, and is known to help with double-stranded repair. Cells of patients with mutations in one of the three cohesin genes have been found to have poor ability to tolerate DNA-damaging agents. Faulty DNA repair regionally could explain the presence of early aging in some body systems, such as the skin, gastrointestinal tract, genitourinary tract, and musculoskeletal system. Long-term effects of disruption of DNA repair could explain many of these clinical finding.

The Mild CdLS Phenotype

A. Cereda, F.J. Ramos, J. Wierzba, G. Gillessen-Kaesbach, S. Maitz, M.P. Ribate, M. Ratajska, J. Limon, J. Pie, Silvia Russo, Lidia Larizza, and A. Selicorni. Classic CdLS needs to be distinguished from the mild CdLS phenotype. As no clear data were available regarding the natural history of this subgroup, we collected clinical and molecular data on a group of patients with a mild CdLS phenotype. We analyzed growth parameters, age of achievement of the main developmental milestones, presence of major congenital malformations, and results of molecular analysis.

We gathered data on 66 mild CdLS patients (30 men; mean age 11 years; range 1–36 years) from Italy, Poland, Germany, and Spain. The mean age of clinical diagnosis was 3 years (range: birth to 25 years). Mean birth weight was 2,514 g (mean gestational age was 38.5 weeks), and a low birth weight (<2,500 g) was found in 39%. Postnatal growth regarding height and weight followed centiles above the 50th centile of dedicated CdLS growth charts in 93.5% and 95% of cases, respectively. No major malformations were present in 36% of patients, and 64% had one or more congenital anomalies (one malformation: 35%; two malformations in 18%; three malformations in 6%; four malformations in 5%). One patient had a limb reduction defect, and two others had ectrodactyly. The major malformations were scored as mild (no treatment needed) in 44% of patients, moderate (nonsurgical treatments) in 18%, and severe (surgical treatments) in 38%.

Mean age of achievement of major developmental milestones were: sitting, 10 months (53%, <9 months; 45%, 9–18 months; 2%, >18 months); walking, 19 months (65%, <18 months; 25%, 18–24 months; 10%, >24 months); first words, 29 months (28%, <18 months; 57%, 18–36 months; 17%, >36 months). Cognitive development was determined to be normal in 9% of patients, borderline in 20%, and mildly delayed in 71%. Molecular analysis showed an NIPBL mutation in 38.5% of patients and an SMC1 mutation in 8%.

These data demonstrated that the mild CdLS phenotype may be less uncommon than previously anticipated. By definition, a normal birth weight, absence of severe major malformations, postnatal growth within normal limits, and a (near) normal or only slightly delayed psychomotor development are the features. Molecular analysis showed the prevalence of NIPBL mutations to be lower compared with the prevalence in series with classical CdLS. Possibly, SMC1A mutations are more common but the series described here are small and ascertainment bias may play a role.

Correlation of Audiological Findings With Clinical Severity and Genotype

P. Marchisio, A. Selicorni, A. Cereda, M. Ceruti, E. Baggi, E. Nazzari, J. Azzollini, C. Gervasini, L. Pignataro, and N. Principi. Hearing impairment is almost universal in CdLS: 20% of CdLS individuals show sensorineural hearing loss (SNHL) and 80% have conductive HL as result of persistent otitis media with effusion (OME). We studied whether there could be a correlation between audiological findings, genotype, and clinical severity as scored by combining auxological, neurodevelopmental, and malformative data.

The study cohort consisted of 44 CdLS children (1–81 years; 22 men). All children received a full otolaryngologic and audiological examination (tympanogram, audiogram, and/or an ABR recording). The diagnosis of OME was based on impaired mobility, opacification, fullness or retraction of the eardrum, associated with a flat tympanogram, and the absence of signs and symptoms of acute infection. Ten children (23%) had SNHL, 26 had conductive HL (59%), and 8 normal hearing (18%). NIPBL mutations were detected in 22 patients (50%): 4 missense mutations, 3 frameshift mutations, 5 splicing site mutations, 8 truncating mutations, and 2 in-frame mutations. No SMC1 mutation was observed. NIPBL mutations were detected in 7/10 children (70%) with SNHL and in 15/34 without SNHL (44%; OR: 2.96). Conductive HL was borderline in 11/26 (42.3%), mild in 10/26 (38.5%), and moderate in 5/26 (19.3%) of the children. The presence of NIPBL mutations did not correlate with the severity of conductive HL. A more severe clinical severity was associated with more marked (≥30 dB) conductive HL (7/8 versus 10/21 with a less marked clinical severity; P = 0.06).

These data showed a correlation between sensorineural HL and NIPBL mutations, possibly explainable by the NIPBL expression in the embryonic inner ear. The association between the severity of conductive HL owing to OME and clinical severity stresses the need for aggressive treatment of OME in CdLS children.

Sensory Input Related to Genotype

Antonie D. Kline, A. Kimball, C. Schoedel, and S. Ishman. CdLS typically presents with a number of malformations and limitations with respect to development. We were interested in assessing sensory deficits in CdLS as these could have a direct effect on learning and on the activities of daily living. We would anticipate that input from the five senses would be diminished and that there would be a genotype–phenotype correlation regarding severity. It has been documented that visual deficits occur, particularly myopia, reported in two-thirds of patients with CdLS, and ptosis, seen in about 40%. In addition, up to 20% have limb truncation defects, and sensory input from a single digit or abnormal limb would be different and difficult physically. Hearing loss in CdLS has been reported in the majority of patients, both conductive and seonsorineural. We reasoned that also anosmia might be present but remain undetected. Anosmia has been associated with other syndromes, such as Down syndrome, Bardet–Biedl syndrome, and other ciliopathies.

We studied 71 adolescent and adult CdLS patients from a multidisciplinary aging clinic, and 12 additional patients evaluated through meetings of the CdLS Foundation. An ophthalmologic evaluation was carried out on each individual. Standard audiology testing was performed on seven patients and records collected on the others. To assess the sensation of smell, the Brief Smell Identification Test™ [Sensonics, Inc., Haddon Heights, NJ] (B-SIT) was performed on those CdLS individuals, able to communicate the answers to us in some way. The Alcohol Sniff Test (AST) was performed on four individuals.

The age range of the participants was 13–50 years with an equal sex ratio. In the aging cohort, 33% were found to have myopia, 20% had high myopia with one secondary retinal detachment, and 42% had ptosis. Both myopia and ptosis were more severe in the individuals with otherwise a more severe phenotype and more frequent in truncating NIPBL mutations. Forty-five percent were found to have some degree of deafness, both sensorineural and conductive. Increased severity of the mutation indicated a worse hearing loss. All 11 patients tested by the B-SIT were able to smell, but the test was only possible in less severely affected individuals. In four more severely affected individuals the AST was performed, and one was unable to smell.

It can be concluded that sensory input is decreased in CdLS, particularly with truncating mutations. Routine audiologic and ophthalmologic evaluations are mandatory in all CdLS patients. Olfactory function seems normal in at least individuals with a less severe phenotype.

Understanding Social Functioning

Joanna Moss, Pat Howlin, Penny Tunnicliffe, Jane Petty, Gemma Griffith, Richard Hastings, Sarah Beaumont, Rachel Yates, and Chris Oliver. Several studies have reported an increased prevalence of autism spectrum characteristics in CdLS. This association cannot be fully accounted for by degree of intellectual disability. The profile of autism spectrum characteristics in CdLS appeared somewhat different from that of idiopathic autism and a presentation of social anxiety has been suggested to be characteristic. We studied the specific nature of social impairments in CdLS, focusing on sociability, motivation for social interaction and stranger discrimination, and compared these with patients with two other syndromes: Cri du Chat syndrome (CdCS) and Angelman syndrome (AS).

Participants were 60 individuals (aged 2–19 years), of whom 20 had CdLS (mean chronological age [CA] = 12.1; SD = 3.4), 20 had CdCS (mean CA = 9.0; SD = 4.8), and 20 AS (mean CA = 10.4; SD = 4.7). The CdCS group scored significantly higher on the Adaptive Behavior Composite score of the VABS-II compared with the AS and CdLS groups (P < 001). The Social Communication Questionnaire (SCQ) and the Sociability Questionnaire for people with Intellectual Disability (SQID) were employed to assess the presence of autistic characteristics and levels of sociability. An observational assessment of sociability, social interaction skills, and stranger discrimination was also conducted. The CdLS group scored significantly higher than the CdCS group on the social interaction subscale and total score of the SCQ (P ≤ 0.01). Significantly more individuals with AS (93.8%) and CdLS (100%) scored above the cut-off for autism spectrum disorder compared with individuals with CdCS (P = 0.001). The difference between the proportion of individuals scoring above the cut-off for autism (CdLS 52.9%; CdCS 13.3%; AS 43.8%) approached significance (P = 0.058). Individuals with CdLS scored significantly lower than those with AS and CdCS on the total familiar score of the SQID (P < 0.02), and significantly lower than individuals with AS on the total unfamiliar score of the SQID (P < 0.02). Analysis of the observational assessments is ongoing but preliminary analyses suggest that our observations are consistent with the profile of scores on the SCQ and SQID.

The findings confirm previous reports of lower levels of overall sociability with both familiar and unfamiliar adults and increased levels of autistic-like characteristics in CdLS compared with individuals with CdCS and AS. These impairments do not appear to be accounted for by degree of intellectual disability and may be considered to be syndrome-specific impairments.

Environmental Determinants of Self-Injurious Behavior

Penny Tunnicliffe, Chris Oliver, Joanna Moss, Jane Petty, Gemma Griffith, Richard Hastings, and Pat Howlin. The prevalence of self-injurious behavior in CdLS is estimated to be approximately 60%. Previous research has identified pain and operant learning as possible causal mechanisms but there have been not been any large-scale studies comparing potential causes across syndrome groups. Additionally, contemporary methods of functional analysis to identify operant learning processes are rarely employed with this group.

We compared the self-injury and aggressive behavior of children aged 2–19 years with CDLS (n = 20; mean CA = 12.1; SD = 3.4), CdCS (n = 20; mean CA = 9.0; SD = 4.8), and AS on an established questionnaire measure (Questionnaire About Behavioral Function; QABF) of the causes of challenging behavior. We also used experimental functional analysis and structured descriptive analyses in which we systematically manipulated environmental conditions in single-case experimental designs.

Analysis of the results of the QABF showed that the CdLS group scored significantly higher than the CdCS and AS groups on the pain subscale (P < 0.02 and P < 0.04, respectively) indicating that pain was related to challenging behavior in this group. Using an arbitrary cut-off of Cliff's d-statistic of .3, within the CDLS group, experimental functional analysis of self-injury revealed an attention-maintained function for four (20%) participants and a demand escape function for six (30%) participants.

The results confirmed previous findings by demonstrating a significant association between pain and challenging behavior in CdLS and the possibility that the behavior is influenced by operant learning for some CdLS children. The comparison with other syndrome groups identified pain as a more prominent cause in CdLS with implications for prioritizing clinical investigation of self-injury in this group.

Issues Related to Feeding

Cheri S. Carrico. Individuals with CdLS exhibit medical, physical, and behavioral challenges that place them at risk for difficulties related to oral feeding. These challenges primarily are associated with a history of gastroesophageal reflux disease (GERD), tube feeding, low muscle tone in the oral area, micrognathia, and cleft palate. A history of autistic tendencies also may be related to feeding difficulties. Additional factors, such as delayed physical growth and possible failure to thrive, may be the result of feeding issues in this population. We studied various aspects of feeding difficulties in 47 CdLS individuals, based on personal observations of feeding behaviors and information reported by caregivers.

Participants included 24 men and 23 women, aged 3 months through 36 years, from countries all over the world. Reported concerns associated with feeding included difficulties in sucking, swallowing, chewing, and biting; consumption of only small bites of food; lack of transition to solid foods; lack of transition to cup drinking; selective eating habits; messy feeding habits; choking, coughing, gagging, vomiting, and spitting food out at meal times; oral defensiveness; crying during meals; making unpleasant faces; “stuffing food;” food aversions, particularly based on texture or taste; and refusal to feed orally. In addition, many individuals required pureed foods and thickened liquids to feed orally, as well as modifications, such as a cup with a spout, for drinking. Most individuals also were reported to be slow eaters. In addition, a history of tube feeding, GERD, and esophagitis were common findings that may be related to the identified feeding challenges.

Primary feeding concerns reported by caregivers included transitioning to oral feeding, promoting eating of solid foods, promoting eating of foods of various textures, encouraging better overall eating habits, and developing appropriate feeding techniques. In addition, although half of the individuals reported good general health, a history of colds, ear infections, sinusitis, fevers, allergies, hoarse voice, asthma, and celiac disease were prevalent.

Individuals with CdLS were found to exhibit a variety of characteristics that lead to challenges in oral feeding: medical conditions, and especially GERD, may make feeding unpleasant; low muscle tone, micrognathia; and cleft palate can make oral feeding difficult; and tube feeding may render oral feeding unnecessary. Individuals who have unpleasant experiences associated with oral feeding or who lack a history of oral feeding frequently develop feeding aversions. Once feeding aversions develop, it is very difficult to encourage oral feeding.

Techniques to facilitate and improve oral feeding vary and depend on the needs of the individual. Oral stimulation should be provided as early as possible, particularly in individuals who are tube fed, to avoid later feeding aversions. Among individuals who cannot tolerate oral presentation of food, non-nutritive tactile stimulation should be provided in the mouth. Taste stimulation, including varying textures and temperatures, should be provided as early as possible. The majority of individuals in this study did not receive feeding therapy. Perhaps with early intervention related to oral feeding, some of the feeding difficulties reported can be minimized.

Molecular Studies of NIPBL, SMC1A, and PDS5A

Cristina Gervasini, Maura Masciadri, Paola Castronovo, Jacopo Azzollini, Donatella Milani, Anna Cereda, Giuseppe Zampino, Angelo Selicorni, Silvia Russo, and Lidia Larizza. The three genes known to be involved in the pathogenesis of CdLS encode for proteins of the cohesin pathway: SMC1A and SMC3 are structural cohesin complex subunits, and NIPBL is a regulatory element. PDS5A is also a gene encoding a regulatory element of the cohesion complex. The essential role for the cohesin complex is to control sister chromatid segregation during mitosis and meiosis and facilitate the repair of damaged DNA. Recent evidence has shown that cohesin binds to the same sites in mammalian genomes as the zing finger transcription factor CTCF suggesting that the developmental deficits of CdLS likely result from dysregulation of gene expression, owing to alterations in the cohesin genes.

We investigated molecularly a sample of 137 Italian CdLS patients. All patients entered a molecular flowchart which implied in sequence the search for mutations of: (i) NIPBL, (ii) SMC1A (for patients negative for NIPBL mutations), and (iii) PDS5A (for patients negative for NIPBL and SMC1A mutations). Mutational screenings for NIPBL, SMC1A, and PDS5A were carried out using DHPLC and direct sequencing. In addition, the NIPBL scan included an MLPA kit to test for large NIPBL exon deletions or duplications. Transcription analysis was performed on carriers of missense, in-frame deletions, and splicing mutations. We identified 42 point mutations (6 missense; 1 in frame deletion; 13 splice-site mutations; 8 nonsense mutations; 13 frameshift mutations; and 1 promoter site mutation). MLPA of NIPBL disclosed five large deletions and one duplication encompassing one or several exons. One patient carrying a deletion affecting NIPBL exons 1–10 was further characterized by FISH analysis and had a 2-Mb deletion which included 14 genes next to NIPBL and indicating a contiguous gene syndrome.

Sixty-seven of the 89 NIPBL-negative patients were tested for SMC1A mutations, which were present in 5: all had point mutations, either in frame deletions or missense mutations. Twenty of the 62 patients negative for NIPBL and SMC1A testing were tested for PDS5A mutations and no mutations were found.

The stepwise molecular analysis enabled us to find the molecular defect in 53 of the 137 patients. The study confirmed the major role of NIPBL in the etiology of CdLS, and the minor role of SMC1A, while thus far no mutation was found in the candidate gene PDS5A. Molecular characterization of CdLS patients is mandatory for an adequate genotype–phenotype correlation study, and our stepwise approach may help in classifying CdLS patients for this purpose.

Exclusion of CTCF Mutations as a Common Cause

David R. FitzPatrick, Cheng Yee Chen, Kathy Williamson, and Stuart McKay. Heterozygous loss-of-function mutations in NIPBL are the most commonly identified molecular lesions in CdLS, accounting for about 40% of cases. Missense mutations in the same gene are generally, but not always, associated with less severe disease. Mutations in SMC1A account for 5% of cases, whereas sequence alterations in SMC3 appear are very rare. The products of these three genes function as structural or loading components of the cohesin complex. The cohesinopathy associated with CdLS is likely to primarily impair gene regulation rather than by impairing chromosome separation at metaphase. A significant proportion of cases with classical CdLS remain unexplained from a genetic point of view. CTCF is a protein that functions as a chromatin “insulator” and it has recently been shown to associate with the cohesin complex in interphase chromosomes. We screened 50 cases of CdLS for mutations in CTCF, located at 16q22.1, but identified no sequence variants, suggesting that mutations in this gene are not a common cause of CdLS. In the course of this study, we also identified four novel nonsense and two novel missense mutations in NIPBL in UK cases. The NIPBL mutations identified in our study together with all published mutations have been submitted to a LOVD mutation database: https://lsdb.hgu.mrc.ac.uk/home.php?select_db=NIPBL

Correlation of Major Malformations With the Genotype

A. Cereda, A. Passarini, C. Gervasini, P. Castronovo, M. Masciadri, A. De Paoli, F. Menni, A. Colli, M.A.Galli, M.A. Pavesi, I. Borzani, and A. Selicorni. Next to limb defects CdLS is characterized by various major malformations although none of them is critical for the clinical diagnosis. We studied the incidence of major malformations in relation to genotype in an Italian CdLS cohort. Only five SMC1A mutations were found which precluded meaningful statistical analysis with SMC1A.

Detailed information on the type and severity of major malformations was gathered in 103 CdLS patients (mean age 14 years; range 1–42 years; 59 males). Ultrasound, radiology, and/or magnetic resonance imaging (MRI) were performed to assess a structural visceral anomaly. Functional renal tests were also performed in all patients.

Kidney malformations were evident in 30% of patients. Creatinine clearance was abnormal in 24% of patients with a structural renal anomaly. NIPBL mutations were detected in 43% of patients with structural renal anomalies, in 22% without a renal malformation, in 57% with an impaired renal function, and in 43% with a normal renal function. Cardiac malformations were demonstrated in 35% of patients, of whom 53% had an NIPBL mutation. In 36% of patients with cardiac defect an NIPBL mutation was found. Heart defects were pulmonary stenosis (28%), atrial septum defects (17%) and ventricular septum defects (19%), and prevalence of NIPBL mutations in these defects were 70% (pulmonary stenosis), 50% (atrial septum defects), and 29% (ventricular septum defects), respectively. Limb reduction defects were evident in 17% of the cohort, of whom 78% had an NIPBL mutation. A CNS anomaly was observed in 32% of those who underwent an NMR: 33% of them had an NIPBL mutation. Genital anomalies were present in 31%, 52% having an NIPBL mutation, and 41% of the patients without genital anomalies had a mutation. Palate anomalies were observed in 13% of patients with 54% having an NIPBL mutation. Major eye anomalies were observed in 4% of the cohort and 50% of them had an NIPBL mutation.

This study confirmed the relatively high frequency of heart and kidney anomalies in CdLS individuals. Decreased renal function was not uncommon and should be evaluated in patients with structural renal anomalies. Analysis of the correlation with the genotype showed a higher prevalence of NIPBL mutations in patients with limb reduction defects, as demonstrated in other studies, and in patients with heart anomalies, especially pulmonary stenosis. For other malformation differences were small and further studies in larger cohorts are needed.

Correlation of Growth and Medical Complications With the Genotype

A. Cereda, A. Passarini, M. Masciadri, C. Gervasini, M. Cerutti, S. Luzzani, F. Macchini, A. Valadè, E. Vismara, and A. Selicorni. Postnatal growth retardation and various medical complications are frequent manifestations of CdLS. We collected detailed information regarding evolution of growth parameters and prevalence of the various medical complications in a cohort of 100 CdLS patients (mean age 14 years; 55 men), whom we also studied molecularly. Postnatal failure to thrive was present in 75% of patients (54% with a NIPBL mutation; in those with normal weight gain, 25% had a mutation). Postnatal growth in height was below the 3rd centile in 84% (54% had an NIPBL mutation; with normal height 30% had a mutation). Microcephaly was present in 77% (44% had an NIPBL mutation; with normal head circumference 22% had a mutation).

The most common medical complication in the CdLS cohort was GER present in 73%. An NIPBL mutation was present equally in cases with or without GER (48% vs. 52%). Behavioral problems were found to be associated with GER: GER was evident in 93% of patients with sleep problems, in 82% of the aggressive patients, but in 30% of hyperactive patients. Seizures were observed in the 26% of the cohort (15% related to fever), of whom 30% had an NIPBL mutation. Blepharitis was evident in 12%, ptosis in 17%, squint in 7%, and refractive errors in 56% (of whom 70% had myopia). NIPBL mutations were present in 43% of those with refractive errors and in 57% of those without.

The prevalence of growth impairments and medical complications in this CdLS cohort was similar to that found in other studies. A NIPBL mutation was more frequent in the patients with markedly decreased growth in height and skull circumference, as found in other studies as well. Data regarding SMC1-mutated patients are limited. Only a tendency for a higher prevalence of seizures in CdLS cases with an SMC1 mutation was clear.

An SMC1A Mutation With Severe Clinical Manifestations

Feliciano J. Ramos, María Pilar Ribate, Milagros Ciero, Juan Carlos de Karam, María Concepción Gil-Rodríguez, María Arnedo, Aliesky Luis Díaz, Beatriz Puisac, and Juan Pié. In about 60% of CdLS patients, mutations in two major genes (NIPBL and SMC1A) of the cohesin complex and its regulators can be found. To date, 11 different mutations in 14 unrelated individuals have been reported in the X-linked SMC1A gene. We investigated a 3-year-old boy with a facial appearance that resembled CdLS only in a limited way, microcephaly, postnatal growth retardation, and moderate psychomotor retardation with complete absence of speech. He had bilateral brachyclinodactyly of the fifth finger and cutaneous syndactyly of the second and third toes. Furthermore, he had congenital heart defects (atrial septum defect and persistent ductus arteriosus) and severe GER causing Sandifer complex with cyanotic, “seizure-like” episodes. He was found to have a novel de novo mutation in the SMC1A gene (p.R711Q) that altered a highly conserved residue and was not detected in his parents or in 50 control individuals. The mutation was located at the SMC coiled-coil domain and may affect the folding of the protein. The patient underscores that, although most reported mutations in SMC1A have caused a mild phenotype, some mutations may result in severe physical and cognitive impairments.

Investigating Large NIPBL Rearrangements

Jolanta Wierzba, Magdalena Ratajska, Jiong Yan, Feng Zhang, James R. Lupski, and Janusz Limon. Most CdLS cases are dominant and sporadic. Approximately half of CdLS individuals carry loss-of-function mutations in NIPBL, about 5% carry an SMC1A mutation, and rarely an SMC3 mutation is found. It has been suggested that patients without mutation in these genes, may carry mutations in other structural components of the cohesin complex (Rad21, Stag2) or mutations could be localized in regulatory sequence for NIPBL.

We studied 11 CdLS patients (9 boys; 4–28 years) who were negative by conventional NIPBL/SMC1A screening (DHPLC and direct sequencing techniques), using multiplex ligation-dependent PCR amplification (MLPA). To confirm different peak patterns, suggesting the presence of large deletion/duplication within these genes, a second MLPA reaction was performed. We found one patient to carry a large deletion within NIPBL. Abnormal RPA ratio was detected for exons 35–47, average RPA reduction was 47%, indicative of a large deletion. The result was confirmed with a second MLPA reaction followed by array-CGH analysis. Array-CGH showed the presence of ∼58-Kb deletion, including the NIPBL and the next FLJ13231 gene. Clinically this patient presented with psychomotor retardation, growth retardation, distinctive facial appearance, and severe gastrointestinal problems.

To our knowledge, this was the second CdLS patient carrying a large NIPBL deletion and the first with a rearrangement extending not only NIPBL but also the neighboring FLJ13231 gene. Large rearrangements in NIPBL are likely rare but may be missed using conventional PCR-based methods. We suggest performing extended mutational analysis in CdLS patients negative for NIPBL/SMC1L1 point mutations.

A Reproductive Risk Questionnaire

Maria Francesca Bedeschi, Vera Bianchi, Faustina Lalatta, Donatella Milani, Anna Cereda, Silvia Maitz, Marta Cerutti, and Angelo Selicorni. CdLS is a genetically heterogeneous disorder, being caused by mutations or deletions in two autosomal genes and one X-linked gene, and sometimes CdLS is the result of an unbalanced translocation which may or may not be familial. Families should be adequately informed about this genetic heterogeneity and the various reproductive risks. We created a questionnaire, to be used as a tool to collect data about the specific needs of CdLS families. The questionnaire consisted of 24 items subdivided in five sections: in Section 1, data were collected about the pedigree and family history; in Section 2, questions about the diagnosis and parents' perception of the disorder were posed; in Section 3, questions were posed about reproductive risks, prenatal diagnosis options, pregnancy surveillance, and parental anxieties; in Section 4, reproductive risk questions for offspring were posed; in Section 5, questions were asked about the parental opinion on genetic testing in general, and also for reproductive counseling and determining the prognosis of their CdLS child and possibly usable in future management. Finally an open question was included, in which parents were asked to provide advice and suggestions for other parents with a CdLS child.

We gathered data from 35 families with 20 male and 15 female CdLS probands (mean age 9.5 years; range 0.9–35 years). Mean age at diagnosis was 1 year (range 0–12 years). In most probands, a moderate to severe developmental delay was reported by the parents. Genetic counseling was offered to 27 of the 35 families. Parents of 19 families considered their reproductive risk regarding CdLS as the same as the risk in the general population. At the time of a subsequent pregnancy 18 of the families asked for a second genetic counseling session. Parents selected as options for prenatal diagnosis amniocentesis and ultrasound scans. The parental opinion about efficacy of prenatal diagnosis was influenced by personal belief and educational status. Usually parents had very limited knowledge and understanding on the limitations of amniocentesis and fetal ultrasounds. Parents showed confidence in genetic testing, to try to define the prognosis of their child, and to apply in future therapies.

Our results showed limited knowledge of the parents about the genetic heterogeneity of CdLS. There is a need for follow-up, to re-evaluate inheritance patterns and risk estimates with the families, and to apply novel knowledge about CdLS in the management.

“You Have to Sit and Explain it All, and Explain Yourself”

Gemma M. Griffith, Richard P. Hastings, Susie Nash, Michael Petalas, Patricia Howlin, Joanna Moss, Chris Oliver, Jane Petty, and Penelope Tunnicliffe. In the United Kingdom, it is estimated that around 50–60% of adults with intellectual disabilities continue to live in the family home well into their middle age. Little research has been conducted about caregivers of adults with intellectual disabilities, and their perceptions of formal support. Some literature suggests that dealings with formal support services may generate further work and effort on the parent's part. However, there is a lack of qualitative family research on adults with rare syndromes, and the purpose of this study was to gather data on the experiences of such families in the United Kingdom.

We used semi-structured interviews to investigate mothers' experiences of formal support. Eight mothers of young adults with either CdLS, AS, or CdCS were interviewed. The interview transcripts were analyzed using Interpretative Phenomenological Analysis. The themes which emerged were: (i) social inclusion and stigma, (ii) uneven medical and social care service provision, (iii) the inertia of social care services, and (iv) mothers as advocates. Parental experiences differed widely across these themes. With day-to-day services, mothers described the benefits of having care staff who were settled in their jobs, whereas others reported multiple problems owing to a high turnover of care staff. In terms of communication, mothers found that formal support services were often indifferent and slow to respond to their enquiries. Generally, mothers reported a lack of awareness of their young adult's syndrome within formal support services, but this did not seem to disadvantage families in terms of their access to formal support. However, it did make a difference to accessing health care, as the rareness of these syndromes are such that medical professionals may be reliant on information parents provide about the syndrome, and as a result may not be able to offer sufficient medical advice to parents.

These findings suggested that accessing appropriate formal support and health services for adults with intellectual disabilities was a lengthy and complex process for parents of adults with rare genetic syndromes. These data may help inform service providers about how best to support young adults with rare genetic syndromes and their carers.

Acknowledgements

The authors thank all the participants who attended the 3rd Cornelia de Lange World Conference for making the event such a success. The Scientific Conference was supported by the Dyna and Fala Weinstock Charitable Trust. They thank all participating Cornelia de Lange Support groups for their continuous support. The work of Dr. Ramos was in part supported by a grant from the Ministerio de Sanidad y Política Social of Spain (Ref. PI061343) and from the Gobierno de Aragón (Ref. B20).

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