Patients
This study included non-surgical HP (ns-HP) patients who visited the endocrinology department of Peking Union Medical College Hospital (PUMCH) from 1975 to 2021. All patients met the diagnostic criteria of HP, hypocalcemia with low or inappropriate normal PTH level. Among 183 HP patients who agreed to conduct gene screening, 36 cases of DGS-1 were detected through TBX1-multiplex ligation dependent probe amplification (TBX1-MLPA) combined with targeted-next generation sequencing (T-NGS), of which 34 cases were available for analysis of CNV. Among the patients who fulfilled the forementioned criteria, those with no rare variants or rare variants classified as benign or likely benign of candidate genes for HP were defined as IHP[9]. Gender and age matched IHP patients were included and analyzed for large CNV, and then the incidence of large CNV and clinical characteristics were compared between patients with DGS-1 and patients with IHP.
Clinical Data
Demographic characteristics, clinical manifestations and treatments of the patients were collected, including gender, age of onset, epileptic seizures, visual impairment, memory impairment, mental impairment, cataract, urinary calculus or calcification, intracranial calcification, Trousseau’s sign, Chvostek’s sign, and the treatment for HP (elemental calcium, active vitamin D and/or high dose of plain vitamin D). Extra-parathyroid manifestations included unusual facial features, slurred speech, repeated infections, arthralgia, renal malformation, hearing impairment, cardiac abnormalities, cleft palate, abnormalities of other endocrine glands, and family history, were also obtained by a chart review of medical records of clinically driven data collection at the clinic of metabolic bone diseases in our center. For patients with suggestive symptoms/signs or family history, it was suggested to the patients or their parents for further evaluation by specialists of other departments (such as slit-lamp by an ophthalmologist, hearing evaluation by otolaryngologist and cardiac evaluation by cardiologist).
Biochemical results at the first and last visit were recorded. Serum total calcium (TCa), ionized calcium (iCa), phosphorus (P), creatinine (Cr), alkaline phosphatase (ALP), potassium (K), magnesium (Mg), total 25-hydroxy-vitamin D (T-25OHD), creatine kinase (CK), total procollagen type 1 N-peptide (TP1NP), β-C-terminal telopeptide of type Ⅰ collagen (β-CTX) were measured with the automatic biochemical analyzer (Beckman Coulter, Indianapolis, IN, USA; AU5800). Twenty-four-hour urine calcium (24hUCa) and phosphate (24hUP) were measured with the automatic biochemical analyzer (Beckman Coulter; AU2700). Serum PTH was measured by chemiluminescence (Siemens ADVIA Centaur, Munich, Germany).
This study has been reviewed by the Research Ethics Committee of PUMCH (JS-3312). All subjects provided informed consent for genetic analysis signed by the patients or his/her parents.
DNA extraction
Genomic DNA was extracted from peripheral blood lymphocytes of subjects with HP using the QIAamp DNA Blood Mini Kit (QIAGEN, Hilden, Germany; LOT 169019868) according to the manufacturer’s protocol.
TBX1-MLPA combined with T-NGS
To screen for large deletions of the TBX1 gene in patients with HP, TBX1-MLPA was performed, using the SALSA® MLPA® Probemix P463-A2 MRKH kit (MRC-Holland, Amsterdam, Netherlands; Lot A2-0520) according to the manufacturer’s instructions as previously described[13].
A gene panel (Agilent Technologies SureSelect™ Target Enrichment System, Santa Clara, CA, USA) was designed to capture all exons and 10-basepair (bp) flanking intron sequences of the 15 candidate genes before 2018[13], including TBX1, GATA3, GCM2, FAM111A, SOX3, TBCE, CHD7, PTH, CASR, GNA11, AP2S1, TRPM6, CLDN16, CLDN19, and AIRE, and 20 genes (5 new genes been added to the list, including NEBL, SEMA3E, DHCR7, HADHA, and HADHB) since 2019, which had been selected from previous studies and the Online Mendelian Inheritance in Man (OMIM) database. T-NGS was performed according to the manufacturer’s instructions.
CNV detection with low-depth Whole Genome Sequencing (WGS)
CNVs (≥ 100kb) was detected by low-depth WGS. The genome DNA of 50 ng was fragmented and DNA libraries were constructed by end fulfilling, adapter ligation, and polymerase chain reaction (PCR) amplification. DNA libraries were subjected to massively parallel sequencing on the NextSeq 500 platform (Illumina, San Diego, CA) to generate approximately 5 million raw sequencing reads with genomic DNA sequences of 36-bp in length. Using the hg19 genomic sequence as reference, a total of 2.8–3.2 million reads were uniquely and precisely mapped using the Burrows-Wheeler algorithm. Mapped reads were allocated progressively to 20-kb bin sizes from the p to q arms of the 24 chromosomes. Counts in each bin were then compared between all test samples run in the same flow cell to evaluate copy number (CN) changes using previously described algorithms. Plots of log2 [mean CN ratio] per bin (Y-axis) versus each 20-kb bin (X-axis) were generated for each of the 24 chromosomes using a blue line to track the mean CNV. For reference, a contiguous blue line running at log2 [0] indicates a CN of 2.0 (disomy) and the dotted lines at position log2 [1.5] and log2 [0.5] indicate the theoretical position of the blue line for a CN of 3.0 (duplication) and a CN of 1.0 (deletion), respectively. For reporting CNV, we applied stringent CN ranges of 2.9–3.1 for a duplication and 0.9–1.1 for a deletion[14].
CNVs identified and mapped are queried and interpreted using publicly available databases, including Decipher, Database of Genomic Variants (DGV), 1000 Genomes and OMIM, such as size, gene content and CNV type (deletion or duplication), and each detected CNV was classified to one of five known categories: pathogenic (P), likely pathogenic (LP), variants of uncertain clinical significance (VUS), likely benign (LB), and benign (B), in accordance with the 2011 recommended guidelines of the International Standard Cytogenomic Array and American College of Medical Genetics and Genomics (ACMG)[15–17]. When necessary, classifications were assessed and revised according to the updated 2020 ACMG guidelines[18]. In cases where variable penetrance and expressivity of a certain CNV has been reported, the CNV was scored as a LP variant.
Statistical analysis
The Kolmogorov-Smirnov test was used to determine the distribution of continuous variables. Normally distributed variables were expressed as mean ± standard deviation (SD) and compared by a Student’s t test. Non-normally distributed variables were expressed as median (interquartile range) and compared by a Mann-Whitney test. Categorical variables were compared by a Pearson χ2 test, Fisher’s exact test, or continuity-adjusted χ2 test. A p value < 0.05 was considered statistically significant. Patients with and without extra-22q11 CNV (≥ 100kb) other than 22q11 deletion were define as CNV positive and negative group, respectively, to compare the clinical data related to HP. The correlation between the size of second CNV burden and clinical phenotype was further analyzed in the group with CNV (≥ 100kb) outside 22q11. In patients with second CNV burden group, the size of CNV was calculated by the sum of base number of CNV other than 22q11. According to gender and age at the first visit to our center, DGS-1 and IHP patients were matched in a 1:1 ratio to DGS-1 patients based on the proportion score with a standard caliper width of 0.2. CNV incidence and clinical characteristics were compared between DGS-1 and IHP patients. All statistical analyses were performed with SPSS 26.0 software (IBM Corp, Armonk, NY, USA).