We report a molecular diagnosis rate of 20.5% in this first Asian Indian KS cohort evaluated by NGS with eight novel P/LP variants in the known CHH genes. ANOS1 was most commonly involved contributing to half of the genetic yield. In this first systematic review of studies (including our cohort) reporting NGS-based multiple gene analysis in KS, the overall molecular diagnosis rate was 30.8% (range: 16.6%-72.2%). The affected genes were FGFR1 (9.6%), ANOS1 (7.7%,) PROKR2 (6.5%), CHD7 (4.6%), oligogenicity (1.9%), FGF8 (1%), SOX10 (1%) and others (PROK2, SEMA3A, IL17RD, GNRHR :1% each). There were geographical differences in the genotype; FGFR1 was predominant in Europe, ANOS1 in India and Brazil, PROKR2 in China whereas CHD7 variants were mostly reported from East Asia.
Molecular diagnosis ranged from 16.6 to 72.2% across centers. As all the included studies in this systematic review had analyzed most of the known CHH genes, the genetic methodology is less likely to account for the variability in molecular diagnostic yield. No increment in genetic yield with the inclusion of additional genetic methods like analysis for copy number variations (CNV) further minimizes the role of genetic methods in the variability of genetic yield . Other attributable factors for varied yield could be variations in cohort size, the proportion of familial cases, and the severity of reproductive phenotype. Notably, studies from (Taiwan, South Korea, and Japan) with a relatively small sample size (n < 20) had a higher yield of 41.6–72.2%, which may be partly due to ascertainment bias [8, 12, 15]. In larger KS cohorts studying adult CHH patients, molecular diagnosis yield was < 20% in China , and India (our center), whereas > 30% in Brazil , and Europe . At our center, the severe reproductive phenotype was associated with a higher molecular diagnostic yield than the partial phenotype (28.3% vs. 4.0%). ANOS1 variants, usually associated with severe CHH , contributed to 50% of the genetically resolved cases in our cohort, which may explain the higher genetic yield in KS patients with severe reproductive phenotype. Similarly a study from China had higher yield (45.6%) likely due to higher percentage of patients with cryptorchidism (a marker of severe phenotype) . Similarly, pediatric cohorts with earlier age at CHH diagnosis due to the frequent occurrence of cryptorchidism reported a higher rate of molecular diagnosis (58.8% and 72.2%) [8, 15]. In contrast, in a few studies with involving patients younger than 18 years without cryptorchidism, the molecular diagnosis rates were relatively low (27.6% and 23.8%) [7, 9]. Although a definitive role cannot be ascertained, ethnic differences in genetic characteristics may account for the variability in the rates of molecular diagnosis among KS cohorts.
Previous studies have demonstrated oligogenic variants in the known CHH genes in up to 20% of patients . In this systematic review analysis, oligogenicity was observed only in 1.9% of probands as it was defined by a stricter definition (molecular diagnosis criteria met individually for two or more genes). Most common gene associated with oligogenicity is FGFR1. Strict adherence to ACMG guidelines should be ensured in ascertaining a pathogenic role for a novel variant and molecular diagnosis or true oligogenicity should be considered only if P/LP variants following the zygosity pattern for known modes of inheritance are observed in a CHH patient.
Our study is the first to highlight the geographical differences in the genetics of KS. Predominantly affected genes were FGFR1 in Europe, ANOS1 in India and Brazil, and PROKR2 in China. Patients with CHD7 variants were mainly reported from East Asia (Japan, China, South Korea, and Taiwan). PROKR2 predominance in the Chinese population can be explained by the presence of a two recurring founder variant (p.Trp178Ser and p.Tyr113His). Despite having non-recurring variants in FGFR1, ANOS1, and CHD7, regional differences observed in their prevalence in KS are probably due to ethnic variation.
Across studies, genetic basis remains unknown in the majority (27.8%-83.4%) of KS. Even after considering VUS following the zygosity pattern as per the known mode of genetic inheritance in a known CHH gene for molecular diagnosis, the genetic yield increased by only 11% (Table 3). Further, the addition of other genetic methods like CNV analysis also did not increase the genetic yield . The genetic cause was not ascertained in 41.6% (5/12) of familial KS probands of our center. This still needs more genetic studies to identify new genetic players in the pathogenesis of KS. Taking all factors into account, a large proportion of KS may not have a monogenic/digenic genetic etiology, and rather may be governed by non-genetic unknown factors.
One of our probands harbored a biallelic P GNRHR variant (p.Arg262Gln) which is not previously described with KS. This proband had partial reproductive phenotype, hyposmia on the clinical history and UPSIT (score: 3/12), and rudimentary olfactory bulbs. The association of this variant in homozygous state with partial reproductive phenotype, but not with hyposmia, has been described previously . We speculate that hyposmia in this proband is due to oligogenicity with a defect in an unknown CHH gene(s) or deletion in a known CHH gene.
Description of molecular diagnosis in Asian Indian KS patients by NGS and systematic collation of NGS-based genetic data of the published KS cohorts is a novel feature of this study. Verification of all the reported variants in the VarSome prediction tool to classify the variants as per ACMG criteria is a major strength of the study (Supplementary Excel). Replication of our previous finding of higher molecular diagnostic yield in severe reproductive phenotype even in KS cohort, similar to nCHH cohort, is noteworthy. A limitation of the review is that some studies could not be included due to a lack of per-patient genotype of KS probands or small cohort size (< 10).
In conclusion, the molecular diagnosis was reached in 20.5% of Asian Indian KS probands with 8 novel P/LP variants, enriching the genetic spectrum of KS. ANOS1 was the most commonly involved gene. The systematic review of NGS-based studies revealed the molecular diagnosis in 30.8% of KS patients; FGFR1 was the most commonly affected gene globally whereas ANOS1 and PROKR2 variants predominated in India and East Asia, respectively. Similar to nCHH cohort, PROKR2 p.Trp178Ser is a recurrent variant in KS cohorts of East Asia. A large number of genetically unresolved KS cases including familial probands warrant further studies with different strategies to unravel the novel molecular mechanisms in them.