A novel 14q13.1–21.1 deletion identified by CNV-Seq in a patient with brain-lung-thyroid syndrome, tooth agenesis and immunodeficiency

Background Chromosome 14q11-q22 deletion syndrome (OMIM 613457) is a rare genomic disorder. The phenotype heterogeneity depends on the deletion size, breakpoints and genes deleted. Critical genes like FOXG1, NKX2–1, PAX9 were identified. Case presentation We performed whole exome sequencing (WES) and copy number variation sequencing (CNV-seq) for a patient with mild speech and motor developmental delay, short stature, recurrent pulmonary infections, tooth agenesis and triad of brain-lung-thyroid syndrome. By using CNV-seq, we identified a 3.1 Mb de novo interstitial deletion of the 14q13.2q21.1 region encompassing 17 OMIM genes including NKX2–1, PAX9 and NFKBIA. Our patient’s phenotype is consistent with other published 14q13 deletion patients. Conclusion Our results showed the combination of WES and CNV-seq is an effective diagnostic strategy for patients with genetic or genomic disorders. After reviewing published patients, we also proposed a new critical region for 14q13 deletion syndrome with is a more benign disorder compared to 14q11-q22 deletion syndrome.

In our study, we report a new patient with 14q13.1q21.1 distal microdeletion syndrome. Copy number variation sequencing (CNV-seq) revealed a de novo 3.10 Mb sized deletion (chr14: 35,268,524-38,367,321). We detailed described this patient's phenotype and reviewed all reported patients with similar breakpoints encompassing NFKBIA, NKX2-1 and PAX9, and provided more information of relationships between clinical features and deleted genes.

Case presentation
The patient is a 15-year-old female born to nonconsanguineous parents by nature labor after full-term gestation. Family history is unremarkable. She was diagnosed as aspiration pneumonia at birth and stayed in hospital for 4 days. Developmental delay was noticed after birth. She could not sit until 12 months. She could speak after 24 months and walk after 27 months. Dysarthria and ataxia were also unnoticed since after. At the age of 2 years, she went to hospital for speech and motor developmental delay. The symptoms were relieved after rehabilitation training. She was also diagnosed as short stature when she was 14 and received growth hormone therapy for 9 months. Her height increased from 132 cm (− 4.7 SD) to 142 cm (− 3.2 SD), resulting in an annual growth velocity of 13 cm.
At the age of 15 years, she was referred to the Department of Respiration, Beijing Children's Hospital for continuous cough and expectoration for 3 years. Her height and weight were below 3rd percentile. She did not have obvious facial anomalies but hypohidrosis and tooth agenesis (missing secondary dentition including molars and premolars) were noticed (Fig. 1a-c). She also had punctate pigmentation was noticed on her back. Mental development was delayed compared to peer children. Immune system examination revealed decreased IgG (3.69 g/L), other immunoglobulins and lymphocyte subsets were normal. Paranasal sinusitis and anemia were diagnosed. She had increased thyroid-stimulating hormone (TSH, 11.983 mIU/L), deceased triiodothyronine (T3, 58.79 ng/dL) and normal tetraiodothyronine (T4, 4.81 μg/dL), indicating a compensated hypothyroidism. She also had delayed development of the secondary sexual characters. Till this visit, she did not have menarche when she was 15. Her bone age was also delayed (BA = 10).
Thoracic computed tomography (CT) revealed interstitial and parenchymal lesions and bronchiectasis in both lungs, dominated with interstitial lesions, and mucus plugs was found in right upper lobe and left lower lobe (Fig. 1d). Ultrasonography of knee joint showed very limited amount of effusion in bilateral suprapatellar capsule. Routine blood test, coagulation function, and screening for infectious diseases, abdominal ultrasonography, ultrasonic cardiography, cranial CT and paranasal sinus CT were all normal.
DNA was isolated from peripheral blood samples obtained from the proband and her parents by using Gentra Puregene Blood Kit (QIAGEN, Hilden, Germany). 200 ng genomic DNA of each individual was sheared by Biorupter (Diagenode, Belgium) to acquire 150~200 bp fragments. The ends of DNA fragment were repaired and Illumina Adaptor was added (Fast Library Prep Kit, iGeneTech, Beijing, China). After sequencing library Fig. 1 Phenotype of the patient. a-b This patient did not have characteristic facial feathers of 14q11-q22 deletion syndrome. c The patient had normal primary dentition and agenesis of permanent teeth. d Thoracic CT showed interstitial and parenchymal lesions and bronchiectasis in both lungs (red arrow) were constructed, the whole exome was captured with AIExome Enrichment Kit V1 (iGeneTech, Beijing, China) and sequenced on Illumina NovaSeq 6000 (Illumina, San Diego, CA) with with 150 base paired-end reads. Raw reads were filtered to remove low quality reads by using FastQC. Clean reads were mapped to the reference genome GRCh37. Single nucleotide variants (SNVs) were annotated and filtered by TGex (http://app. genecards.cn) and classified following the American College of Medical Genetics and Genomics and the Association for Molecular Pathology interpretation standards and guidelines [9]. To identify large copy number variations (CNVs), part of the sequencing library was sequenced directly on Illumina NovaSeq 6000 and each sample yielded one Gigabase Raw data. An in-house pipeline was applied to map and call CNVs and the health parents were used as control samples [10]. Clean reads were mapped to the reference genome GRCh37. Database of Genomic Variants, DECIPHER database, ClinVar, OMIM and ClinGen were used for interpretation and classification of the clinical significance of candidate CNVs according to previously reported guidelines [11]. By whole exome sequencing (WES), 30,636 SNVs and small indels were called from 14,549.59 Mb Clean bases (Target mean depth was 122.23X) in the proband. After analysis and interpretation, none of these variants could explain the proband's phenotype. However, CNVseq revealed a de novo 3. was not present in the parents. WES reads depth data was used to validate this deletion (log2 = − 1.00428, mean depth = 50.02X, probes number = 379). Informed consent was obtained from the parents of the patient.

Discussion and conclusions
In this study, we described clinical and molecular findings in a female with mild speech and motor developmental delay, short stature, recurrent pulmonary infections, tooth agenesis and triad of BLTS. Using CNV-seq, we identified a 3.1 Mb de novo interstitial deletion of the 14q13.2q21.1 region encompassing 17 OMIM genes.
The deletion region of our patient (chr14: 35,268,524-38,367,321) encompasses RALGAPA1 and NFKBIA but no NPAS3 or SEC23A. Consequently, joint laxity, HPE or ACC was not observed in our patient. Epilepsy was also absent in our patient even though RALGAPA1 was deleted. We reviewed published ten patients with phenotype description and similar deletion regions encompassing RALGAPA1, the deleted sizes ranged from 0.82 Mb to 6.98 Mb, and only one patient with 1.99 Mb deletion reported by Caliebe A et al. had seizures [4,5,8,19,23,[27][28][29]. Therefore, deletion of RALGAPA1 was not sufficient to cause seizures and the genotype-phenotype correlation of RALGAPA1 deletion remained unclear. Considering our patient had decreased IgG, paranasal sinusitis and recurrent infections, we also reviewed immunological features of seven patients with NFKBIA deletion (two patients were collected from DECIPHER database, Fig. 3). Patient reported by Villafuerte B et al. also had low IgG but also relatively low lymphocyte count and percentage of switched B cells [8]. Gentile M patient also had recurrent infections, with a mild reduction of CD3/CD8 lymphocytes and an elevation of CD4/ CD8 ratio, yet her IgA, IgG, and IgM were normal [4]. Santen G patient 5 and Peall K patient 4 had recurrent lower respiratory infections [5,19]. Patient 256,879 from DECIPHER database also had recurrent infections. We next reviewed seven patients with PAX9, NKX2-1 deletion and leaving NFKBIA intact in previously literature. The deleted sizes ranged from 0.36 Mb to 3.69 Mb, and only one male with 2.34 Mb had recurrent bronchitises [5,17,19,20,29]. It was notable that the pLI (probability of LoF intolerant) value of NFKBIA was 1, and the o/ e score is 0 (0-0.19), indicating deletion of this gene may have serious clinical consequences. In addition, three NFKBIA nonsense variants were reported in patients with EDAID2 [30]. To this respect, NFKBIA haploinsufficiency may be an appropriate explanation of the immunological features of 14q13 deletion patients.
Our patient, together with previously reported patients, identified a well-defined, more benign, 14q13 distal microdeletion syndrome. The major phenotype includes choreoathetosis, tooth agenesis, pulmonary dysfunction, immunological abnormal and hypothyroidism. The 1.4 Mb critical region contained at least three candidate genes: NFKBIA, NKX2-1 and PAX9 (Fig. 3). In this region, one more gene, PSMA6, with its pLI being 1 and o/e score being 0 (0-0.23), should be more attention. PSMA6 encodes the component of the 20S core proteasome complex involved in the proteolytic degradation of most intracellular proteins. Variants in PSMA6 were reported to be associated with inflammation diseases like myocardial infarction [31], arthritis [32,33], etc. Given its essential function and intolerant of LoF variants in population, more subtle features may be uncovered in the future.
Previous researches have demonstrated that CNV occurred in 5-10% of the total human genome [34], and chromosomal microarray analysis (CMA), as a stable and accurate platform, is used for detecting. Currently, Fig. 3 Schematic representation of the chromosomal region deleted of our patient (blue) and other similar deletions at 14q13.2-q21 described in the previously reported literature (green) and DECIPHER database (black). Three critical genes were located in the overlapping region CNV-seq is developed by analyzing data generated from WES or whole genome sequencing (WGS) [35][36][37]. Recent studies showed that the combination of WES and CNV-seq by low cover genome sequencing increased diagnostic yield in patients with rare diseases [38,39]. In our study, after suspecting the diagnosis of BLTS of our patient, we performed trio-WES and low coverage WGS (0.3X) simultaneously. No putative pathogenic variants in NKX2-1 was identified, but we uncovered a 3.1 Mb deletion encompassing NKX2-1, as well as PAX9 and NFKBIA. Notably, WES data could be applied to CNV identification, so we used WES data to validate this deletion. By reviewing our deleted region and other reported patients, we proposed a 1.4 Mb critical region for 14q13 distal microdeletion syndrome with a well-defined, more benign phenotype compared to 14q11-q22 deletion syndrome. Our results further demonstrated the clinical utility of the diagnostic strategy combining WES and CNV-seq for genetic diseases patients.

Acknowledgements
We thank the patient and the parents for their participation.
Authors' contributions XH drafted the manuscript and analyzed WES and CNV-seq data. RG and JG performed molecular experiments. JL and ZZ performed the clinical examination of the patient and his family. WL performed data interpretation and reviewed the manuscript. BX and CH managed the patient and critically revised the paper. All authors read and approved the final manuscript.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
The study was approved by the Ethics Committee of Beijing Children's Hospital, Capital Medical University.

Consent for publication
Informed consent was obtained from the parents of the patient.