Population demographics

The mean age of the couples who participated in the program was 30.53 years, and the median age was 30 years. Of the 1,512 southern and southwestern Chinese couples, the majority came from Hunan and Guizhou, China, with 700 and 561 couples, respectively. The sample sizes of Guangxi and Guangdong were smaller (209 and 42 pairs, respectively); therefore, our study had a total of 996 pairs of southern couples and 561 pairs of southwestern couples (Supplementary Fig. S1). Since our questionnaire was not mandatory for all participants to complete, we obtained only 2,890 (95.57%) questionnaire results, the details of which are available in Supplementary Table S4. Among the couples who provided information from the questionnaire, excluding three who did not want to disclose their ethnicity, 16 different Chinese ethnic groups were included. Han individuals had the largest number of subjects with 2,501 (82.71%), whereas ethnic minority individuals accounted for a relatively small number of 386 (12.76%). However, it is worth noting that although there were fewer subjects from ethnic minorities, there was a sizable proportion of ethnic minorities among the subjects in the Guizhou and Guangxi regions. There were 212 (54.92%) and 69 (17.88%) participants, respectively, and the minority samples in these regions accounted for a large proportion of the total sample of participants in their regions (18.89% and 16.51%, respectively).

Evaluation of Quality Metrics

In our cohort, all samples were subjected to capture enrichment and sequencing of the exonic and nearby regions of target genes. In all samples, the average mapped_reads was 15,811,070, and the mapped_reads rate was over 99.7%. The average sequencing depth of the target region was 504.81, the average target region coverage (1×coverage) was 99.94%, and the proportion of target region depth ≥ 20X sites was 99.76% on average. Detailed QC information is provided in Supplementary Table S2.

Carrier status of recessively inherited diseases in the Southern and Southwestern Chinese individuals

Among the 220 recessive genes, 931 were classified as pathogenic or likely pathogenic (P/LP; Supplementary Table S3). Candidate variants were found in 1885 subjects, and 2837 P/LP variants were reported, with an average of 0.938 P/LP variants per subject (no chimeras were found in our subjects). More than half of our subjects (62.3%) were carriers of at least one P/LP variant of a recessively inherited disease, that is, the P/LP variant carrier frequency was 62.3%. It was found that 703 (23.2%) were carriers of multiple recessively inherited diseases, of which 535 (17.7%) were carriers of two recessively inherited diseases, 136 (4.5%) were carriers of three recessively inherited diseases, 23 (0.8%) were carriers of four recessively inherited diseases, and nine (0.3%) were carriers of five recessively inherited diseases. (Table 1). Since our subjects were couples, we also counted the carrier status of the P/LP variants in each couple. Almost half of these couples were ‘1 + 0’ carriers (one of the couples was a carrier and the other was a negative individual) (47.7%, n = 721 couples), 211 couples were normal (both were negative individuals) (14.4%), and 580 were ‘1 + 1’ type carriers (both couples were carriers of recessive P/LP variants) (38.4%) (Table 2).

Table 1 Estimated burden of carriers in the study cohortTable 2 Estimated burden of couples with different carrier status in the study cohort

The carrier frequencies for the top 15 most common conditions are listed in Table 3. In the Southern and Southwestern Chinese populations, the carrier frequencies of 10 conditions exceeded 2%, and two conditions exceeded 5%, including Deafness, Autosomal Recessive1A (DFNB1A) (n = 643, 21.3%), and α-thalassemia (n = 224, 7.4%). GJB2-associated DFNB1A is a recessive disease with the highest carrier frequency among all diseases, far exceeding that of other diseases. It was also three times higher than that in α-thalassemia (n = 224, 7.9%) and seven times higher than that in Wilson’s disease (n = 96, 3.4%). It is worth noting that nine of the 15 recessively inherited diseases with the highest carriage frequencies were metabolic system diseases; as such, metabolic system diseases should be considered essential in the ECS panel.

Table 3 Top 15 conditions with the highest carrier rates in the Southern and Southwestern Chinese population

The top 10 most common variants in the southern and southwestern Chinese populations and the percentage of these variants in the total variants of their genes are shown in Fig. 1. The most common variant in our cohort was GJB2 c.109G > A p.V37I, which was associated with DFNB1A and was found in 557 subjects (18.4%). A total of eight variants had a carrier frequency higher than 1%, with two of the HBA1/HBA2 having a carrier frequency higher than 2%, and both were in the top five of the carrier frequency, -α3.7 (3.1%) and -SEA (2.3%), respectively. Among the 10 most common variants, the following accounted for the majority (> 50%) of all variants detected in this gene: GJB2 c.109 (n = 557, 86.6%), POLG c.2890 C > T (n = 79, 97.5%), SMN1 exon7del (n = 57, 100%), GALC c.1901T > C (n = 55, 74.3%), SLC25A13 c.852_855del (n = 40, 64.5%). In addition, the two previously mentioned variation loci HBA1/HBA2 -α3.7 and HBA1/HBA2 -SEA together also accounted for the majority in HBA1/HBA2 (n = 163, 72.8%).

Fig. 1

Top 10 most common P/LP variants and the total number of variants in the corresponding genes in the Southern and southwestern Chinese populations. The data in the bar graph show the number of the top 10 most common P/LP variants, and the data in the dotted line graph show the total number of variants in the corresponding genes for these variants. The lower X-axis represents the top 10 most common P/LP variants, and the upper X-axis indicates the genes corresponding to the variants

Analysis of test results for specific diseases

In our cohort, some specific diseases could not be screened for variations by NGS. We identified these variations by TP-PCR combined with capillary electrophoresis or LR-PCR combined with gel electrophoresis; as such, these results have been further separately analysed. Of the 3024 individuals screened, none carried intron 1 or intron 22 variants of the F8 gene. A total of 1512 women were screened for FMR1 CGG repeat length; none of them had > 200 CGGs (pathogenic variant carriers), two (0.13%) had 55–200 CGGs (premutation carriers), and 12 (0.79%) had intermediate (45–55 CGGs) (Supplementary Table S5). For women carrying premutation mutations, not every pregnancy occurs with an increase in the copy number of CGG triplet repeat sequences, but all premutation mutations are at risk of a large increase in copy number to become full mutations during inheritance; therefore, we provided risk alerts for these two subjects.

Comparative analysis of carrying frequencies between the Southern and Southwestern regions of China

We categorized 3024 subjects into 1122 from southwestern China and 1902 from southern China, according to their detailed geographic locations. We analyzed the detected variants by category and the results are presented in Table 4. First, the carrier frequency and number of variants per subject in southwestern China were slightly higher than in southern China; however, they were almost the same. In terms of the top 10 most common conditions, there were subtle differences between the southern and southwestern regions. In these 10 conditions, the sum of the frequencies in the southwestern part of China was not much different from that in the southern part, which is 51.9% and 47.1%, respectively, suggesting that recessively inherited diseases carried by the subjects in both the southern and southwestern regions of China are not dispersed. Although the top two diseases carried in both regions were GJB2-associated DFNB1A and HBA1/HBA2-associated Alpha-thalassemia, the frequency of both in the southwest was approximately 2% higher than in the south. Of interest were ATP7B-associated Wilson’s disease and HBB-associated β-thalassemia, which were present at considerably higher frequencies in the southwestern subjects than in the southern subjects. In contrast, individuals in Southern China had significantly higher frequencies of mutation carriage for POLG-associated Autosomal recessive progressive external ophthalmoplegia and CYP21A2-associated classical congenital adrenal hyperplasia due to 21- hydroxylase deficiency than those in southwestern China. This suggests that the carrier statuses in southern and southwestern China have their own characteristics, with certain genes having significantly higher carrier frequencies than in the overall population.

Table 4

Top 10 conditions with the highest carrier rates in southern China and southwestern China, respectively.Diseases with large differences in carrier frequencies between southern and southwestern China were targeted using different colors

Comparison to previously published carrier frequencies

We compared the carrier frequencies of the top 10 most common diseases in southwestern China and southern China obtained from our CS with those from previous CS studies, and the results are shown in Table 5 (the literature relied upon to derive the expected carrier frequencies of the Top 10 most common conditions can be found in Additional File S2). The expected carrier frequencies were derived from the combined predictions of previous CS studies, and after a chi-squared goodness-of-fit test, the observed carrier frequencies were statistically different from the expected carrier frequencies for the six diseases. The disease with the largest difference in carrier frequency was HBA1/HBA2-associated α-thalassemia, which nearly doubled in frequency. In addition, eight of the top 10 most common diseases listed in Table 4 had observed carrier frequencies greater than the expected carrier frequencies. Only two diseases, GJB2-associated DFNB1A and G6PD-associated glucose-phosphate dehydrogenase deficiency, had observed carrier frequencies that were slightly lower than the expected carrier frequencies, and there was no statistically significant difference between the observed and expected carrier frequencies of GJB2.

Table 5 Top 10 conditions with the highest observed carrier frequencies with corresponding observed carrier frequency and expected carrier frequencyAt-risk couples and affected theoretical pregnancies

When both individuals carry variants of the same recessive gene, the likelihood of giving birth to a child with a recessive disease is as high as 25%. A total of 210 (6.94%) individuals from all the southern subjects carried the same autosomal recessive genes as their partners. However, we excluded some couples from the ARCs because of the relatively smaller adverse risk to the offspring when they jointly carried the GJB2 c.109G > A variant, as described in the methods. Thus, there were 51 pairs of autosomal recessive ARCs (3.44%), of which 18 pairs were deafness-associated (GJB2), 19 pairs were thalassemia (HBA1/HBA2 or HBB)-associated, and 15 pairs were other systems (cases 22G00284093 and 22G00284094 were both deafness-associated ARCs and other systems ARCs). In addition, there were 77 female subjects carrying X-linked recessive gene variations who were at risk of causing the birth of an affected son (1/4) (cases 23G00040615 and 23G00040616 were both thalassemia-associated ARCs and X-linked recessive disease-associated ARCs). Thus, our ARCs totaled 127 ARCs (51 autosomal recessive and 77 X-linked recessive pairs, with one pair having both autosomal recessive and X-linked recessive disease-associated ARCs). Of the 127 ARCs, 47 (19 autosomal recessive and 28 X-linked recessive) were southwestern couples, and 80 (32 autosomal recessive and 49 X-linked recessive) were southern couples (one couple had both autosomal recessive and X-linked recessive disease-associated ARCs) (Supplementary Table S6). The frequencies of ARCs in southern and southwestern China were 8.38% and 8.41%, respectively, indicating that there was little difference in the frequencies of ARCs between southern and southwestern China. In all the subjects, the theoretical number of offspring affected without intervention was 2.10%.

Follow-up results and evaluation of the effectiveness of ECS application

After all the reports were distributed to the subjects, we followed up with 1512 couples in three groups, preparation for pregnancy, pregnancy, and postpartum, to study the effectiveness of ECS. We received 1,422 follow-ups, with 1,031, 263, and 128 couples in the three groups, respectively, for a follow-up rate of 94.1%. Among the nonpregnant couples, 409 (39.7%) had no current pregnancy plans, 597 (57.9%) chose natural conception to have offspring, and 25 (2.4%) switched from natural conception to preimplantation genetic testing for monogenic (PGT-M). Of the pregnant couples, 245 chose to continue the pregnancy, 10 couples had spontaneous abortions, and eight couples chose induced abortions (due to the detection of chromosomal disorders or monogenic disorders). Of the 14 pregnant couples with fetal abnormalities (detected by ultrasound, non-invasive, and other prenatal tests), nine couples were tested for genetic abnormalities, and four couples had positive results. Among the postnatal couples, 64 (50%) underwent postnatal genetic testing, of which seven had positive results. Fortunately, six of these couples had newborns who were carriers of the causative gene rather than patients, and the remaining couple had a newborn with GJB2, c.109G > A purity who passed the hearing screening.