Cri-du-Chat Syndrome: Revealing a Familial Atypical Deletion in 5p

Abstract

Introduction

Cri-du-chat syndrome is generally diagnosed when patients present a high-pitched cry at birth, microcephaly, ocular hypertelorism, and prominent nasal bridge. The karyotype is useful to confirm deletions in the short arm of chromosome 5 (5p–) greater than 10 Mb. In cases of smaller deletions, it is necessary to resort to other molecular techniques such as fluorescence in situ hybridization, multiplex ligation-dependent probe amplification (MLPA) or genomic array.

Case Presentation

We report a family with an atypical deletion in 5p (mother and 2 children) and variable phenotypes compared with the literature. We applied a P064 MLPA kit to evaluate 5p− in the mother and the 2 children, and we used the Infinium CytoSNP-850K BeadChip genomic array to evaluate the siblings, an 11-year-old boy and a 13-year-old girl, to better define the 5p breakpoints. Both children presented a high-pitched cry at birth, but they did not present any of the typical physical features of 5p− syndrome. The MLPA technique with 5 probes for the 5p region revealed that the patients and their mother presented an atypical deletion with only 4 probes deleted (TERT_ex2, TERT_ex13, CLPTM1L, and IRX4). The genomic array performed in the siblings' samples revealed a 6.2-Mb terminal deletion in 5p15.33p15.32, which was likely inherited from their mother, who presented similar molecular features, seen in MLPA.

Discussion

The sparing of the CTNND2 gene, which is associated with cerebral development, in both siblings may explain why these 2 patients had features such as better communication skills which most patients with larger 5p deletions usually do not present. In addition, both patients had smaller deletions than those found in patients with a typical 5p− phenotype. This report demonstrates the utility of genomic arrays as a diagnostic tool to better characterize atypical deletions in known syndromes such as 5p− syndrome, which will allow a better understanding of the genotype-phenotype correlations.

Established Facts

• The main clinical feature of cri-du-chat syndrome (CdCS) is the high-pitched cat-like cry in newborns.

• Karyotype analysis is the only test available for most Brazilian patients with clinical manifestations of CdCS.

Novel Insights

• We report the investigation of a familial 6.2-Mb atypical deletion in 5p.

• We highlight the importance of performing cytogenomic tests for precise delineation of 5p deletion breakpoints to improve genotype-phenotype correlation.

Introduction

Cri-du-chat syndrome (CdCS; OMIM #123450) is a rare genetic syndrome caused by partial or total deletion of the short arm of chromosome 5 (5p–) with an incidence ranging from 1:15,000 to 1:50,000 live births. The main clinical features of CdCS are a high-pitched cat-like cry in newborns, low birth weight and growth delay, microcephaly, facial asymmetry, epicanthal folds, hypotonia, ocular hypertelorism, low-set ears, prominent nasal bridge, micrognathia, and developmental delay, including difficulties in mobility, dexterity, and verbal communication [Niebuhr, 1978; Mainardi et al., 2006; Nakagami et al., 2015].

Although genomic arrays are a first-tier diagnostic test for patients with developmental disabilities and/or congenital abnormalities, karyotypes are the only test available for most Brazilian patients with clinical manifestations of CdCS, and deletions smaller than 10 Mb remain undetectable.

For atypical cases, fluorescence in situ hybridization (FISH) can be used to check for chromosomal translocations that involve the 5p region, and multiplex ligation-dependent probe amplification (MLPA) and genomic arrays have greater resolution and specificity to identify which genes are affected by a given chromosomal deletion. Diagnosis with these technologies may be delayed due to the availability of this molecular testing, mainly in developing countries [Nguyen et al., 2015; Yokoyama et al., 2018].

Herein, we report the analysis of a familial 6.2-Mb atypical deletion in 5p using a combination of MLPA and genomic array for 2 siblings and MLPA for their mother that revealed the same terminal deletion and emphasized the importance of mapping 5p regions to better establish genotype-phenotype correlations in 5p− syndrome.

Case Reports

We studied 3 individuals from the same family: a mother and her 2 children, a 13-year-old daughter and an 11-year-old son. The mother also had a third child who did not have intellectual disability or any of the clinical features presented by the other 2 siblings.

At evaluation at the age of 13 years, the girl weighed 65 kg (97th centile) and was 156 cm (44th centile) tall. She was the product of the first pregnancy of her nonconsanguineous parents. There were no complications during childbirth, and she was full-term. She had no difficulty in gaining weight and took her first steps at the age of 2. The clinical findings included high-pitched cry at birth, intellectual disability, 56 cm head circumference (98th centile − 13 years), high palate, clinodactyly, prominent nasal bridge, bitemporal narrowing, short philtrum, syndactyly type I-a, sandal gap, and rounded face (Fig. 1).

Fig. 1.

Facial (a) and lateral view (b) of the girl at the age of 12 years. Facial features include a prominent nasal bridge and short philtrum.

At evaluation at the age of 11 years, the boy weighed 23 kg (<3rd centile) and was 124 cm (<3rd centile) tall. He was the product of the mother's second pregnancy with the same partner. There were no complications during childbirth, and he was born at 9 months. He had difficulty in gaining weight and took his first steps at 3 years of age. The clinical findings included high-pitched cry at birth, intellectual disability, 51 cm head circumference (5th centile − 11 years), failure to thrive, craniofacial asymmetry, strabismus, dysplastic ears, high palate, clinodactyly, prominent forehead, bilateral epicanthal folds, plagiocephaly, scoliosis, triangular face, and autistic traits (Fig. 2). All features for both siblings are described in Table 1.

Fig. 2.

Facial (a) and lateral view (b) of the boy at the age of 10 years. Facial features include strabismus and prominent forehead.

Table 1.

Comparative table of the clinical findings in patients with 5p– in this study and in 3 other reference studies

Features	Niebuhr, 1978 (331 patients)	Mainardi et al., 2006 (159 patients)	Elmakky et al., 2014 (5.5-Mb deletion in twins)	Present study (6.2-Mb deletion)
				girl (at evaluation)	boy (at evaluation)
Microcephaly	279/288	NA	+	–	–
Ocular hypertelorism	196/252	105/129	+	–	–
Low-set ears	146/208	81/116	–	–	–
Micrognathia	136/189	119/123	–	–	–
Hypotonia	67/143	78/108	+	–	–
High-pitched cat-like cry	267/284	141/147	+	+	+
Epicanthal folds	223/252	119/132	+	–	+
Prominent nasal bridge	248/261	102/117	+	+	–
Low birth weight	86.9%	NA	+	–	+
Growth delay	66.7%	NA	+	–	–
Intellectual disability	295/296	NA	–	+	+
Developmental delay	NA	NA	–	+	+
Autistic traits	NA	NA	–	–	+
Craniofacial asymmetry	NA	NA	–	–	+
Strabismus	87/156	48/101	+ (for the girl)	–	+
Dysplastic ears	101/123	NA	–	–	+
High palate	48/86	62/74	+	+	+
Clinodactyly	38/75	NA	–	+	+
Prominent forehead	NA	NA	–	–	+
Bitemporal narrowing	NA	NA	–	+	–
Short philtrum	NA	52/86	–	+	–
Plagiocephaly	NA	NA	–	–	+
Syndactyly	36/116	NA	–	+	–
Scoliosis	NA	20/47	–	–	+
Sandal gap	NA	NA	–	+	–
Rounded face	124/183	96/115	+	+	–
Triangular face	NA	NA	–	–	+
Synophrys	NA	NA	+	–	–
Pointed chin	NA	NA	+	–	–
Single palmar creases	NA	NA	+	–	–

NA, information not available; +, presence of the characteristic; -, absence of the characteristic.

Both siblings form simple sentences, communicate using gestures, understand commands, and know colors, letters, and numbers. However, neither sibling can read nor write. Both siblings are able to eat and perform personal hygiene without assistance.

The mother was evaluated briefly since she appeared to the clinical evaluation only once with her children; all the subsequent appointments were arranged with the children's father. She can read and write but does not work outside her house. Information about whether she finished high school was unavailable. The father did not appear to have any intellectual or other clinical problems, and he finished high school with no higher education.

Taking into consideration the classification of the level of intellectual disability proposed by Zhang et al. [2005], the degree of cognitive delay estimated for the siblings was moderate and that for the mother was mild.

Materials and Methods

Subjects

The healthy father and the 2 children were followed by geneticists at the Unit of Clinical Genetics, Instituto da Criança, Hospital das Clínicas, Universidade de São Paulo (ICr-HCFMUSP), Brazil.

DNA Extraction

Genomic DNA was isolated from peripheral blood lymphocytes using a commercially available DNA isolation kit (QIAamp DNA Blood Mini Kit®, Qiagen) according to the manufacturer's instructions. The quality and quantity of the DNA samples were determined using a Qubit® Fluorometer (Invitrogen), and the integrity of the DNA was evaluated via agarose gel electrophoresis analysis.

MLPA

The MLPA assay was performed using the P064 Microdeletion Syndromes-1B kit (MRC Holland®), which includes probes for the main microdeletion and microduplication syndromes as well as 5 probes for the 5p region. DNA denaturation, hybridization of probes, ligation, and PCR were performed in accordance with the manufacturer's instructions. The separation of amplified products by electrophoresis was performed using an ABI 3500 Genetic Analyser (Applied Biosystems®), and the data were analyzed using GeneMarker® software, version 1.6 (www.softgenetics.com). The peak area of each fragment was compared to that of a control sample, and the results were considered abnormal when the relative peak-height ratio was less than 0.70 (deletion) or greater than 1.30 (duplication). The details of the regions detected by each kit can be found at www.mlpa.com.

Array

A genomic array was performed for the siblings using the Infinium CytoSNP-850K BeadChip, which contains approximately 850,000 selected single nucleotide polymorphisms with enriched coverage for 3,262 disease-related genes (Illumina, Inc.). The raw data were analyzed using BlueFuse^TM Multi software, version v4.5 (BlueGnome®). The genomic positions are presented as mapped to the GRCh37/hg19 genome build.

The results were analyzed according to the American College of Medical Genetics and Genomics (ACMG) guidelines [Riggs et al., 2020] and following databanks of copy number variation (CNV) and classified as benign, likely benign, variant of uncertain significance (VUS), likely pathogenic or pathogenic: the Database of Genomic Variants (DGV, http://projects.tcag.ca/variation/), the Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources (DECIPHER, http://decipher.sanger.ac.uk/), and the UCSC Genome Bioinformatics database (http://genome.ucsc.edu).

Results

We investigated the family with 2 children presenting moderate intellectual disability. The children's samples were analyzed by the MPLA technique for detection of the main microduplication and microdeletion syndromes, and the results revealed a 5p deletion that supported the hypothesis of CdCS for both children.

Upon knowing the positive result for 5p–, we realized that both siblings presented the same deleted probes, implying that the deletion may have been inherited from one of the parents; therefore, the parents were invited to also be tested.

The MLPA results from the parents revealed that the mother carried a 5p deletion while the father had no alteration in 5p. An array technique was used to determine the genomic breakpoints for both siblings.

Cytogenetic Results

A G-banded karyotype was performed before MLPA for the parents and children in order to investigate the possibility of a rearrangement between chromosome 5 and other chromosomes, but the results showed a normal karyotype for all the investigated subjects (the 6.2-Mb deletion was not detected in any karyotype). A repetition of G-banding was done after MLPA and array results to confirm that the 5p terminal deletion was not visible.

MLPA Results

We detected deletion of 4 probes in 5p15 (TERT_ex2, TERT_ex13, CLPTM1L, and IRX4) in the mother and in the siblings using a P064 kit (Fig. 3). The P064 kit contains 5 probes in 5p, and we observed that only 4 were deleted, characterizing an atypical deletion. The CTNND2 probe was not deleted.

Fig. 3.

MLPA of the mother (a) and daughter (b) showing deletion of 4 probes from 5p (TERT_ex2, TERT_ex13, CLPTM1L, and IRX4, respectively) highlighted in red color.

Array Results

For the siblings, the results showed the presence of a terminal deletion encompassing 5p15.33p15.32 (Fig. 4), and the precise positions of the deleted probes encompassed coordinates 25,328–6,189,781 bp for the boy and 25,328–6,197,044 bp for the girl. The 25,328-bp position represents the position of the first probe from the BeadChip, indicating the most terminal probe in 5p.

Fig. 4.

Array of the brother. This image indicates the deleted region in the short arm of chromosome 5 identifying the 6.2-Mb deletion.

We observed a slight difference between the final breakpoints in both siblings (one probe difference). Most likely both deletions in the siblings are the same size and these breakpoint differences might be due to some reasons such as improper hybridization during array or a fluorescent signal misinterpreted by the BeadChip scanner (Illumina Assay Guide). Although there is a small breakpoint difference, analysis in genetic databases showed that this difference indicates no relevant clinical difference between both siblings.

Discussion

The phenotypic variability of CdCS has been associated with the heterogeneity of the deletion size, position, and gene content at the short arm of chromosome 5. The deletions vary in size, but breakpoints have been mainly described within bands 5p15.2 or 5p15.3 [Chehimi et al., 2020].

A critical region for the syndrome between 5p15.2 and 5p15.3 has been defined by Nguyen et al. [2015], who classified the following 5 genes as being haploinsufficient and related to the phenotype in patients with CdCS: TERT, SEMA5A, MARCH6, NPR3, and CTNND2. Hemizygosity of the TERT gene has been implicated in the dysfunction of telomere-length maintenance, and the deletion of SEMA5A may disrupt normal brain development. The deletion of MARCH6 plays a role in the development of facial features and dysmorphisms, and deletions of the NPR3 gene have been implicated in the associated cardiovascular diseases [Wu et al., 2005; Duan et al., 2014; Santo et al., 2016].

The CTNND2 gene, which was not deleted in the subjects of the present study, is associated with neuronal development/function and cellular death. The loss of a copy of CTNND2 in CdCS may be associated with intellectual disability, reading problems, and learning difficulties [Hofmeister et al., 2015; Corrêa et al., 2019]. Medina et al. [2000] observed the phenotype in patients with 5p deletion: hemizygous deletion of the CTNND2 gene resulted in moderate to severe intellectual disability, while patients without the deletion of CTNND2 demonstrated mild to moderate intellectual disability. Furthermore, Sardina et al. [2014] observed mild intellectual disability in a unique female with CdCS and partial deletion/partial duplication of the CTNND2 gene. They propose that partial duplication of the CTNND2 gene, including the promoter, may mitigate the cognitive impairment in individuals with 5p deletion.

The siblings investigated here can form simple sentences, communicate using gestures, and understand commands, but can neither read nor write. The siblings can understand other people, and such receptive language skills are usually not present in CdCS. The sparing of the CTNND2 gene may explain why the siblings have fewer communication issues. The intellectual development of the mother was not assessed since she showed up only once in her children's appointment and was not properly evaluated.

One patient in the DECIPHER database (314863) has a deletion similar to that found in the siblings, and information provided in the database explains that the deletion was paternally inherited, with no clarification whether it is a pure terminal deletion or a balanced translocation. However, there is no information about his or his parents' phenotype. Inheritance in 5p− has been rarely documented.

Elmakky et al. [2014] described a 3-generation family with an unbalanced whole-arm translocation between chromosomes 5 and 15 as well as a terminal microdeletion of 5.5 Mb involving 5p15.33p15.32. The probands were Caucasian male and female twins, and they had common features of 5p− as follows: high-pitched cry, small round face, epicanthal folds, microcephaly, and broad nasal bridge. However, the probands also had features that are not typical for 5p− as follows: synophrys, pointed chin, and single palmar creases. At a visit at 24 months, neurological development was normal for both. All clinical features of the probands are listed in Table 1.

With regard to the present cases, the boy is introspective, and he does not have a good relationship with classmates at school. He presents receptive language and expressive language. He is irritable, aggressive, and has autistic traits. The girl does not have similar behavioral features. We did not find any CNV related to autism spectrum disorder (ASD). There are several genes that may cause ASD in the literature, but studies on the relationship between ASD and CdCS are still lacking [Wang et al., 2009].

The girl is in the 98th centile for head circumference, which places her on the threshold for macrocephaly according to Marinescu et al. [2000]. This observation differs from the findings in the literature for CdCS where the typical feature is microcephaly. Therefore, her head circumference is a rare feature.

In early CdCS reports, the genotype-phenotype correlation was limited by the use of available research techniques, such as G-banding and FISH, but this analysis has been refined with the advancement of molecular genome analysis methodologies. The main advantage of the array technique is the ability to investigate the entire genome in a single experiment with higher resolution and accuracy when compared to the FISH and MLPA techniques. The main limitations of the array technique are the high cost of large-scale application for developing countries and the need for a specialized team to analyze the results [Zhang et al., 2005; Vallespín et al., 2013; Zanardo et al., 2017].

A complete evaluation and different investigative tools, such as checklists (evaluation of family background as well as physical and intellectual development of the patient), can lead to the most accurate path in the cytogenomic investigation to clarify the genetic cause of intellectual disability.

Determining the mechanism leading to 5p− is essential for counseling and recurrence risk estimation. Most 5p deletions are de novo (80–90%), and approximately 10% of 5p deletions are inherited from a balanced translocation carrier, usually the father [Liu et al., 2017; Su et al., 2019]. In this family, the mother had the 5p deletion and passed it on to her 2 children, which highlights the importance of genetic counseling for the family.

There is still a need to clarify the phenotypic differences found in the siblings. Epigenetic and environmental factors may also affect phenotype and hamper genotype-phenotype correlation, indicating that DNA methylation should also be studied. DNA methylation alters gene expression without modifying the DNA nucleotide sequence [Archer et al., 2011; Naumova et al., 2017], therefore, the methylation status of the genes in 5p and elsewhere could be explored as possibly contributing to intrafamilial variability.

One example of the possible influence of methylation in 5p− clinical presentation was given by Marignier et al. [2012] who reported an 11-year-old girl with a 5p deletion of 12.85 Mb, encompassing CTNND2, with apraxia of speech but with normal intellectual performance (Fig. 5). As previously mentioned by Hofmeister et al. [2015] and Corrêa et al. [2019], CTNND2 has been classified as haploinsufficient and associated with intellectual disability, so with this finding, it would be interesting to explore in future investigations its contribution to the phenotypes in this syndrome.

Fig. 5.

Representation of the extent of the deletions, indicated by the horizontal lilac bars, and breakpoints in basepairs detected by array. The genes are marked with vertical arrows. 1 and 2 represent the siblings; 3, 4, and 5 are the cases from the literature discussed in this manuscript with different proposed critical regions for indicated features. The cited features and corresponding references are included at the bottom of the scheme. Adapted from Chehimi et al. [2020].

Another 3-generation family study reported that the female proband with 5p− (10.5-Mb deletion) had psychotic symptoms at the age of 62 years. Disability status examination indicated that she had moderate intellectual disability, auditory hallucinations, delusions of persecution, hot temper, self-talking, self-laughing, and aggressive behavior. These behaviors are not typical for CdCS. Haploinsufficiency of the dopamine transporter gene (SLC6A3) at 5p15.3 has been proposed to be associated with behavioral problems in CdCS, which may be linked with psychiatric manifestations [Fang et al., 2008]. While this family had a typical CdCS 5p deletion, this behavior is not typical. This leads us to think about possible age effects in CdCS. However, it is very rare to have information about individuals with 5p− at advanced ages.

Fang et al. [2008] also reported intrafamilial variability. The affected females had more serious behavioral problems, such as more severe intellectual disability and prominent psychotic symptoms, whereas the male patients had less severe intellectual disability with no psychotic symptoms. While this previous report has more severely affected females, in our family, the brother is the one who has the more severe behavioral symptoms. So, the sex bias would not be consistent. Genetic modifiers and/or environmental interactions have been proposed to explain intrafamilial variability [Cirillo et al., 2014].

Conclusion

In this work, we describe a rare case of a familial 6.2-Mb atypical deletion in 5p and present the cytogenomic analysis. We highlight the importance of performing cytogenomic tests for precise delineation of 5p deletion breakpoints to improve genotype-phenotype correlation, and we emphasize the importance of genetic counseling in families with chromosomal imbalances. In addition, the study of other epigenetic mechanisms in future investigations can contribute to clarify the genotype-phenotype correlation in this syndrome.

Statement of Ethics

The study was approved by the Ethics Committee for Analysis of Research Project HCFMUSP/CAPPesq (CAAE: 24390719.1.0000.0068). Written informed consent was obtained from the parent/legal guardian of the patients for publication of the details of their medical case and any accompanying images.

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

This work was supported by FAPESP (Fundaçao de Amparo à Pesquisa do Estado de Sao Paulo) grant number 2018/02385-0.

Author Contributions

V.T. Almeida wrote the paper and performed cytogenomic analysis. S.N. Chehimi performed cytogenomic analysis and genotype-phenotype correlations. A.M. Nascimento prepared the samples and performed DNA extraction. Y. Gasparini and G.F.S. Carvalho discussed the results. M.M. Montenegro, E.A. Zanardo, and A.T. Dias discussed the molecular and clinical results of the article. N.A. Assuncao was responsible for the funding sources. C.A. Kim provided the samples and clinically assessed the patients. L.D. Kulikowski designed and coordinated the study. All authors read and approved the final manuscript.

Data Availability Statement

The data that support the findings of this study are openly available in https://figshare.com/articles/dataset/Array_girl/16786147 and https://figshare.com/articles/dataset/array_boy/16786135.

Funding Statement

This work was supported by FAPESP (Fundaçao de Amparo à Pesquisa do Estado de Sao Paulo) grant number 2018/02385-0.