A detailed survey was conducted of all family members. The data were recorded and sorted according to the phenotypes of each member. Representative photos of the phenotype were obtained from some family members. Peripheral venous blood for DNA extraction was collected into evacuated EDTA tubes from most members of the family. Genomic DNA was prepared from peripheral blood leukocytes. This study was approved by the Zhujiang Hospital Ethics Committee, an affiliate of the Southern Medical University (Guangdong, China).

Karyotyping was performed on phytohemagglutinin-stimulated peripheral blood lymphocytes according to standard clinical procedures. Karyotypes of the family members were analyzed using G-banding chromosome analysis (Zhang et al., 2020). The karyotypes were described according to the International System for Human Cytogenomic Nomenclature standards (ISCN 2016). Partial cell suspensions were stored at −20°C.

To further understand if minor deletions were present on the chromosomes, comparative genomic hybridization experiments (array CGH) were performed on five samples using the Affymetrix Cytoscan® 750 k (Affymetrix, Santa Clara, CA, United States). This platform includes 200,000 SNP markers and 500,000 copy number variation (CNV) markers that are distributed across the human genome at an average density of about 1 marker/4 kb (Zhang et al., 2020). The arrays were strictly run according to the manufacturer’s protocol and the data were visualized and analyzed using the Chromosome Analysis Suite software package (Affymetrix). The February 2009 human reference sequence (GRCh37/Hg19, http://genome.ucsc.edu/) was used as the reference sequence.

Primers were designed and the duplication or deletion of chromosome fragments was verified in different patients through PCR. To verify the balanced translocation through PCR analysis, two sets of primers were designed for the chromosome where the translocation occurred. PCR was performed with specific oligonucleotide primers as follows: chromosome 4 binding primer F: 5′- GTC​CCG​GTC​CAT​AAC​GCT​TGC -3′ and R: 5′- GGA​AGT​TAT​GTG​TAC​CGG​AT -3’ (amplified fragment length of 420bp); chromosome two binding primer F: 5′- GAT​CAT​TAC​CGA​GTC​TTT​CTG -3′ and R: 5′- CGT​CTG​GGT​TGC​CAC​AGA​CGA -3’ (amplified fragment length of 550 bp). The PCR products were analyzed by electrophoresis in 2% agarose gel, followed by ethidium-bromide staining.

The proband was a 25-year-old woman who went to the hospital for medical examination due to ID. No abnormalities were found on electroencephalogram tests. It was learned through interviews that this phenomenon also existed in some of the family members of this woman’s family. Among the family members, three had similar physical characteristics as the proband. These included tall, overweight, signs of dementia on the face, duck steps, and ID. In contrast, seven other family members had short, underweight statures, stunted growth, and were unable to restrict their behavior. The pedigree of this family is shown in Figure 1, while the detailed phenotypic records of family members are shown in Table 1. The differences among healthy, deletion, duplication, and translocation patients are shown in Table 2.

Among the surviving family members, the clinical details of the patients with more obvious symptoms are as follows:

IV-10: This 25-year-old female measured 163 cm in height, weighed 90 kg (overweight), and presented with small eyes, a drooping upper left eyelid, a full moon face, wide palms, lack of joints between the ring finger and metacarpal, a short ring finger, and large feet (wearing size 44 shoes on the European scale). Born at full term (40 weeks), she weighed 5 kg at birth with good post-natal feeding. Facial reaction dementia was noted 6–7 months after birth, but no abnormalities were found in the hospital following an EEG examination and other tests. Her intelligence test value was low. While she can take care of herself and communicate with people, she often gives irrelevant answers to questions. She was illiterate and lacked numeracy skills. She walks like a duck (Figure 2A).

IV-14: This 24-year-old female measured 157 cm in height and weighed between 90 and 95 kg. She weighed 3.2 kg at birth. She is married and was currently pregnant in utero for 7 + months (V-6). Except for typical hand bones and joints, the remaining clinical manifestations were the same as IV-10 (Figure 2B).

IV-15: This 17-year-old male measured 130 cm in height and weighed 25 kg (underweight). Born at full-term (40 weeks), he weighed 4 kg at birth. His suction was diminished, he ate less, and he developed slowly. The hospital diagnosed the patient with congenital malnutrition. While this patient is active, he is not good at communicating with others, is unable to pronounce words clearly, and cannot restrict his behavior. He was illiterate and had no numeracy skills (Figure 2C).

IV-18: This 13-year-old male measured 110 cm in height, was underweight, had big eyes and double eyelids, and a pointed chin. He weighed 2 kg at birth, had poor suction, ate less frequently when compared to the norm, and was stunted in growth. No abnormalities were seen during the hospitalization. While this patient is active, he cannot restrain his own behavior, is unable to pronounce words clearly, is not good at communicating with others, and often provides irrelevant answers to questions. He had no literacy or numeracy skills. His body posture and clinical manifestations were the same as for patient IV-15 (Figure 2D).

In order to determine whether the patients of this family had intellectual disabilities due to chromosomal abnormalities, blood samples of some of the family members (patients and healthy control samples) were subjected to chromosome analysis. The results showed that the karyotypes of III-3, III-4, III-6, III-7, III-8, III-9, III-12, IV-7, IV-10, IV-11, IV-14, IV-15, IV-18, and IV-19 were normal.

To further understand if there were minor deletions on the chromosomes, comparative genomic hybridization experiments (array CGH) were performed on five samples. The proband (IV-10) and IV-14 were found to harbor the same CNVs, with a 2.2 Mb 2q terminal deletion on chromosome two and a 4.6 Mb 4p terminal duplication on chromosome 4. The latter included the entire 4p16.3 region and part of 4p16.2. However, IV-18, who has a different phenotype, was found to have the opposite CNVs with a 2.2 Mb 2q terminal duplication noted on chromosome two and a 4.6 Mb 4p terminal deletion on chromosome 4. Besides there being a difference in length of less than 10bp, the deletion and duplication positions were the same as IV-10 and IV-14. The CNVs of chromosomes two and 4 of the above-mentioned patients originated from the same balanced translocation (Figure 3A). In addition, IV-10 not only possessed a 2.2 Mb 2q terminal deletion on chromosome two and 4.6 Mb 4p terminal duplication on chromosome 4, but also a 411 kb Xp duplication on her X chromosome. Among the family members, III-6 and IV-11 are in good health without any evidence of dysplasia. Except for congenital heart disease found in IV-11, all remaining family members are healthy.

Based on the CGH results, it was speculated that a balanced translocation was present in the family members. To verify this, two sets of primers were designed for the chromosomes where the translocation occurred (Figure 3B). The forward and reverse primer binding positions of the first set of primers were for 2q and 4q respectively, that is, they could only bind to chromosome 4 with 2q fragments. The second set of primers were constructed in a similar way in that 4p fragments would bind to chromosome 2. The two sets of primers are used to amplify the patient’s DNA. Banding noted on gel electrophoresis confirmed the presence of the translocation, while an absence of bands indicated normal chromosomal structure. Combining the two sets of results indicated the presence or absence of the translocation of chromosomes 2 and 4 in family members (Figures 3C,D).