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Keywords:

  • 1p36 deletion;
  • 22q11.2 deletion;
  • cardiomyopathy;
  • chromosomal anomaly;
  • non-compaction left ventricle

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Non-compaction of the left ventricle (NCLV) is a cardiomyopathy characterized by prominent left ventricular trabeculae and deep intertrabecular recesses. Associated extracardiac anomalies occur in 14–66% of patients of different series, while chromosomal anomalies were reported in sporadic cases. We investigated the prevalence of chromosomal imbalances in 25 syndromic patients with NCLV, using standard cytogenetic, subtelomeric fluorescent in situ hybridization, and array-comparative genomic hybridization (CGH) analyses. Standard chromosome analysis disclosed an abnormality in three (12%) patients, including a 45,X/46,XX mosaic, a 45,X/46,X,i(Y)(p11) mosaic, and a de novo Robertsonian 13;14 translocation in a child affected by hypomelanosis of Ito. Cryptic chromosome anomalies were found in six (24%) cases, including 1p36 deletion in two patients, 7p14.3p14.1 deletion, 18p subtelomeric deletion, 22q11.2 deletion associated with velo-cardio-facial syndrome, and distal 22q11.2 deletion, each in one case. These results recommend accurate clinical evaluation of patients with NCLV, and suggest that chromosome anomalies occur in about one third of syndromic NCLV individuals, without metabolic/neuromuscular disorder. Array-CGH analysis should be included in the diagnostic protocol of these patients, because different submicroscopic imbalances are causally associated with this disorder and can pinpoint candidate genes for this cardiomyopathy.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Non-compaction of the left ventricle (NCLV) is a recently defined cardiomyopathy characterized by prominent left ventricular trabeculae and deep intertrabecular recesses. Genetic etiology is highly heterogeneous, including sporadic or familial isolated non-syndromic NCLV (NS-NCLV) resulting from single gene mutations, and syndromic NCLV (S-NCLV) associated with genetic disorders, metabolic and neuromuscular diseases [1, 2]. Chin et al. [1] first noted distinct facial features associated with developmental delay in three patients with NCLV, and suggested that this disorder could be part of a genetic disorder. The frequency of extracardiac anomalies in patients with NCLV is ranging between 14% and 66% in different series [1, 3, 4]. Chromosome anomalies have been discovered in sporadic cases, including one fourth of the patients with 1p36 deletion [5], 1q43 deletion (associated with terminal 4q deletion in one case) [6], 5q35 deletion [7], 8p23.1 deletion [8], trisomy 13 [9], trisomy 18 [10], 22q11.2 deletion [4], distal 22q11.2 deletion [11], and Turner syndrome [12]. S-NCLV and NS-NCLV loci have been found on chromosome Xq28 (Barth syndrome), 3p22.2, 10q23.2, 11p11.2, 14ptel-14q12, and 18q12.2 [3, 13, 14].

In this study we have investigated the frequency of chromosome anomalies in a consecutive series of S-NCLV, using standard cytogenetic, subtelomeric fluorescent in situ hybridization (FISH) and array-based comparative genomic hybridization (array-CGH) analyses.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Fifty-six patients with NCLV have been evaluated consecutively at the Bambino Gesù Pediatric Hospital and La Sapienza University in Rome, between January 2003 and December 2011. Echocardiographic criteria for diagnosis of NCLV included: (i) presence of multiple echocardiographic trabeculations; (ii) multiple deep intertrabecular recesses communicating with the ventricular cavity, as shown by color Doppler imaging and the recesses showed in the apical or middle portion of the ventricle; and (iii) two-layered structure of the endocardium with a non-compacted to compacted ratio >1.4 [15]. Two-dimensional color-Doppler echocardiography was performed also in the patients' parents.

Accurate clinical examination was performed, in order to check for the presence of major and minor associated anomalies. Family history was collected.

Standard chromosome analysis at 550-band resolution and subtelomeric FISH using ToTel Vysion-kit (Vysis Inc., Downers Grove, IL) according to manufacturer's suggestions were carried out on phytohemagglutinin-stimulated circulating lymphocytes in syndromic patients. Array-CGH analysis was performed using a genomic oligonucleotide-array with an effective resolution of 250 kb (Human Genome Microarray 4 × 44K Chip; Agilent Technologies, Walldbronn, Germany). Genomic DNA was extracted from venous blood using a commercial kit (Macherey-Nagel, Düren, Germany) and was hybridized against a reference DNA (Promega, Madison, WI). The arrays were imaged with Feature Extraction software (v9.5.1) and chromosomal profiles were obtained using the ADM-2 algorithm provided by DNA Analytics software (v4.0) (Agilent Technologies). Copy number changes were confirmed by locus-specific FISH, using commercial probes (Vysis Inc.) or bacterial artificial chromosome clones selected from a genomic library (32K library; BACPAC Resources, Oakland, CA). DNA was extracted by Quantum Prep MiniPrep Kit (BioRad, Hercules, CA) and SpectrumGreen-dUTP or SpectrumOrange-dUTP labeled using the Nick Translation kit (Vysis Inc.), according to the manufacturer's protocol. Metaphase spreads were obtained following standard procedures and FISH analysis was performed as described in Bernardini et al. [16].

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Among the 56 NCLV patients, 29 (52%) were NS-NCLV and 27 (48%) syndromic. S-NCLV patients included 15 males (56%) and 12 females (44%), with an age at time of genetic evaluation ranging from 1 month to 31 years (mean age ± SD 5.1 ± 2.7). Ethnicity was Caucasian in all patients. Two syndromic patients had Barth syndrome, and were excluded from cytogenetic analysis. Clinical and molecular-cytogenetic results of the 25 syndromic patients are summarized in Table 1.

Table 1. Clinical, cardiac, and genetic characteristics of 25 patients with syndromic NCLV
NSexClinicalGenetic diagnosisCardiac defect diagnosisAge at cardiac diagnosis (years)MRFacialExtracardiac anom.Other malformationsPrevious reports
  1. Ao, aortic; Asc, ascending; ASDos, ostium secundum atrial septal defect; CGH, comparative genomic hybridization; co, coarctation; del, deletion; F, female; ins, insufficiency; IUGR, intrauterine growth retardation; LSVC, left superior vena cava; M, male; MR, mental retardation; N, number; NCLV, non-compaction of the left ventricle; PDA, patent ductus arteriosus; t, translocation; ter, telomeric; VCF, velo-cardio-facial; VSD, ventricular septal defect; +, present feature, −, absent feature, +/−, mild expression.

1FTurner syndrome45,X(10%)/46,XX (90%) de novoNCLV6.1++Short stature [12, 18]
2MSyndromic45,X(70%)/46,X,i(Y)(p11) (30%) de novoNCLV4.5+
3FHypomelanosis Itot (13;14) de novoNCLV, subaortic and muscular VSD1.7++Linear cutaneous acromic lesions, IUGR, medial facial angioma, second to third toe syndactyly
4Fdel 1p36 syndromedel 1p36 mother with negative array-CGH, father not availableNCLV, muscular VSD, PDA0.4++Epilepsy, feeding difficulties [4, 5, 19-21]
5Fdel 1p36 syndromedel 1p36 de novoNCLV11.2++Epilepsy, feeding difficulties [4, 5, 19-21]
6FSyndromicdel 18pter de novo (mother with del 3q29)NCLV4.7++Esophageal atresia, otodysplasiaShort stature, deafness
7FDiGeorge/VCFdel 22q11.2 de novoNCLV, muscular VSD1.1+Laryngeal web [4, 17]
8FSyndromicdel 7p14.3p14.1 mother with negative array-CGH, father not availableNCLV, muscular VSDs ASDos, Ao valve dysplasia0.1++Sacral fistulaIUGR, growth difficulties, microcephaly
9MSyndromicDistal del 22q11.2 de novoNCLV, bicuspid aortic valve, muscular VSD, persistent LSVC0.9+/−+Single umbilical arteryGrowth difficulties, hypotonia, microcephaly, umbilical hernia[11]
10FMarfan syndromeArray-CGH negativeNCLV, Asc ao dilatation, Bicuspid ao valve5.2+Tall stature, arachnodactyly, scoliosis[22]
11MNoonan syndromeArray-CGH negative PTPN11 gene negativeNCLV0.6+[23]
12MLEOPARD syndromePTPN11 gene mutation (Tyr279Cys) array-CGH negativeNCLV7.4+Lentigines[24]
13MCornelia-de Lange syndromeArray-CGH negativeNCLV, Ao co0.8++Growth retardation[25]
14FCHARGE syndromeArray-CGH negative CHD7 gene mutationNCLV, ASDos3.3++Ocular colobomaSeventh cranic nerve palsy, deafness
15MSepto-optic dysplasiaArray-CGH negativeNCLV0.7+Septo-optic dysplasiaShort stature
16MBardet–Biedl syndromeArray-CGH negativeNCLV8.3+Postaxial poly-dactyly hand and foot, foot syndactylyObesity
17MSyndromicArray-CGH negativeNCLV, ASDos4.6++Corpus callosum agenesis, cryptorchidismShort stature
18MSyndromicArray-CGH negativeNCLV8.3++Cerebellar hypoplasiaRenal tubular acidosis
19MSyndromicArray-CGH negativeNCLV0.8+Hepatic cavernoma
20MSyndromicArray-CGH negativeNCLV0.1++Laryngomalacia, macroglossiaMicrocephaly, feeding difficulties, growth deficiency
21MSyndromicArray-CGH negativeNCLV, Ao valve dysplasia, bicuspid ao valve, mitral ins, mitral dysplasia1.7++Pre-axial polydactylyGrowth deficiency
22FSyndromicArray-CGH negativeNCLV, muscular VSD, long Q-T0.5++
23FSyndromicArray-CGH negativeNCLV, polyvalvular dysplasia0.8++Cleft palate, feet anomaliesGrowth deficiency
24MSyndromicArray-CGH negativeNCLV1.3++Hypotonia
25FSyndromicArray-CGH negativeNCLV4.7++Hand anomalies

Cardiac evaluation

Characteristic non-compaction morphology was limited to the left ventricle in 24 syndromic cases, while affected both ventricles in 2. NCLV was associated with a congenital heart defect (CHD) in 12 of 25 (48%) cases. Additional cardiac anomalies are shown in Table 1. Two patients underwent cardiac transplantation (patients 11 and 25 in Table 1).

NCLV was sporadic in 25 families, while recurrence of S-NCLV in two male siblings born to unrelated unaffected parents was found in one family (patient 17 in Table 1). Two-dimensional color-Doppler echocardiography was normal in parents of 24 probands. The mother of patient 10 had Marfan syndrome with dilatation of the ascending aorta without NCLV.

Clinical evaluation

Clinical features are summarized in Table 1. Electromyography was performed in patients 8, 9, 18, 20, and 24 (Table 1), and the examination was normal in all of them.

Family history disclosed: recurrence of NCLV with corpus callosum agenesis, psychomotor delay, and similar facial anomalies in the brother of patient 17; recurrence of mental retardation, cerebellar hypolasia, and facial anomalies without NCLV or renal tubular acidosis in the male cousin of patient 18; an unaffected dizygous co-twin of patient 9; segregation of Marfan syndrome from the mother of patient 10.

Molecular and cytogenetic results

Standard chromosome analysis was normal in 23 of 25 (92%) patients (Table 1). One girl was a 45,X (10%) /46,XX (90%) mosaic, one male patient was a 45,X(70%)/46,i(Y)(p11)(30%) mosaic, while one patient with a clinical diagnosis of hypomelanosis of Ito was a de novo 13;14 translocation heterozygote. In this subject parental disomy was excluded. Cryptic chromosome anomalies were diagnosed in six (24%) patients. Subtelomeric FISH analysis confirmed the diagnosis of 1p36 deletion in two cases (patients 4 and 5), and disclosed subtelomeric 18p deletion in patient 6. FISH analysis with N25 probe confirmed 22q11.2 deletion in patient 7. FISH analysis with a probe specific for the SRY locus confirmed Yp duplication in patient 2. Array-CGH analysis disclosed a 7p14.3-14.1 deletion spanning for about 6.3 Mb of genomic DNA from 34,904,091 to 41,246,668 Mb (based on hg18 release) in patient 8, and distal 1.6 Mb deletion 22q11.2, spanning from 20,256,261 to probe 21,933,489, in patient 9 (hg18).These two imbalances were not listed among copy number variants in public database (http://projects.tcag.ca/variation/). Patients with gross chromosome anomalies (patients 1, 2 and 3) and the patient with 22q11.2 deletion (patient 7) disclosed no additional anomalies at array-CGH analysis.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

This study on a consecutive series of patients with S-NCLV has disclosed chromosome anomalies in about one third of the cases. Different numerical and, mainly, structural defects have been found, the majority of which were cryptic imbalances. The recurrence of NCLV in some specific chromosomal anomalies suggests a causal-effect relation.

It should be considered that the so-called NCLV is an intriguing cardiomyopathy which is a source of controversies in the literature. In fact, it is questionable if it is really a pathologic entity or simply an anatomical variant of normal. In this regard, it is possible that the NCLV appearance may be one of the various phenotypic expression, probably innocent, of several syndromes, and not necessary a really nosographic entity.

Nevertheless, some cryptic chromosomal imbalances seems to have a specific relationship with NCLV. For example, the link between 1p36 deletion syndrome and NCLV is well established, occurring in 23% of the cases [5]. This association was corroborated by two patients in the present series.

Deletion 22q11.2 was associated with NCLV in two previous patients [4, 17]. Deletion 22q11.2, distal to the classical DiGeorge syndrome deletion, has been reported as a new genomic disorder. A possible association between distal deletion 22q11.2 and NCLV has been recently reported [11], and confirmed in a second patient in this study. Interestingly, anatomic heart characteristics in the Madan's [11] patient and in our case 9 are similar, consisting of NCLV associated with muscular ventricular septal defect and bicuspid aortic valve, with dilatation of the aortic root and ascending aorta as a possible cardiac marker for the disease.

Two cases at least of Turner syndrome with NCLV are known (12, present case), and the male patient in our series carrying a mosaic X monosomy in 70% of blood cells, associated with a derivative Y chromosome with Yp duplication and loss of Yq in 30% of the cells, can also be added among sex chromosomal anomalies associated with NCLV. It is possible that the patient with ‘Turner mosaic and male phenotype’ described by Altenberger et al. [18], with a mos45,X/46,X,+mar/47,X, +2mar karyotype, could be considered a similar case. At best of our knowledge, 7p14.3-14.1 and 18p deletions have not been associated previously with this heart anomaly.

Additional CHDs, associated with NCLV, were documented in more than half of the cases (5/9 = 56%) in the present series of chromosomally unbalanced patients.

In conclusion, accurate clinical evaluation is required in patients with NCLV, in order to detect associated extracardiac anomalies. A great variety of chromosome imbalances occurs in about one third of S-NCLV patients, prompting a systematic array-CGH analysis in these cases. Hopefully, this strategy will also provide some insight into the localization of new candidate regions or genes for NCLV.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References