Highlights
-
We report atypical and rare alleles affecting the HTT gene, including extremes of repeat lengths, biallelic expansions with clinical correlates.
-
One-sixth of cases lacked family history of HD, though some had late-onset psychiatric symptoms, suggesting low-pathogenic expansions.
-
Three novel CAG expansions transmitted from intermediate alleles highlight intergenerational instability and the need for family-based studies.
Introduction
Huntington’s disease (HD) is a progressive neurodegenerative disorder clinically characterized by a spectrum of motor, behavioral and cognitive symptoms. HD is caused by a triplet (CAG) repeat expansion in exon 1 of the Huntingtin (HTT) gene on chromosome 4 [Online Mendelian Inheritance in Man (OMIM) 613004] and is inherited in an autosomal dominant manner.Reference Macdonald, Ambrose and Duyao1 Alleles with CAG expansions > 39 are fully penetrant (FPA), while alleles with 36–39 repeats are called reduced penetrance alleles (RPA) as they may or may not develop any symptoms in their lifetime. Alleles with repeats between 27 and 35 called intermediate alleles (IA) do not cause HD but are highly unstable and prone to pathogenic expansions (>35) in the offspring.Reference Bean and Bayrak-Toydemir2 The length of the CAG repeat inversely correlates with age at onset (AAO) of motor symptoms.Reference Andrew, Paul Goldberg and Kremer3 Other genetic and environmental modifiers can also modify the AAO and the outcome.Reference Wexler, Lorimer and Porter4 The prevalence of HD, at-risk haplotypes, IA and the incidence of de novo mutations seem to vary across populations, suggesting possible differences in the extent of CAG instability.Reference Warby, Montpetit and Hayden5–Reference Ruiz De Sabando, Urrutia Lafuente and Galbete9 Previous studies have reported on CAG instability during transmission and the prevalence of latent risk of HD posed by IAs and RPAs.Reference Myers, MacDonald and Koroshetz10,Reference McNeil, Novelletto and Srinidhi11 Despite reports suggesting that HD is not uncommon in India, there is sparse data on the borderline expansions and their transmission trends across generations. We analyzed a well-characterized family-based cohort and report the demographics, inheritance patterns, instability trends and pertinent clinical correlates.
Materials and methods
Study design and cohort
This was an ambispective observational study conducted at the National Institute of Mental Health and Neurosciences, Bengaluru, India. A total of 390 individuals underwent genetic testing for HD between 2011 and 2019. Of these, 239 tested positive for expanded CAG repeats in the HTT gene (Figure 1). Twelve individuals lacked sufficient clinical information, and seven were presymptomatic gene-positive individuals with diagnostic confidence level (DCL) = 0 on the UHDRS total motor score (TMS). These were excluded from age-at-onset analysis, yielding a final cohort of 220 symptomatic individuals. The study was approved by the Institutional Ethics Committee (Ref No. NIMH/DO/ETHICS SUB-COMMITTEE 29th MEETING/2016), and written informed consent was obtained. Pre- and post-test genetic counseling was provided to all individuals or their caregivers.
Overview of the clinical categorization and genetic testing outcomes of 390 individuals evaluated during the study period. IA = intermediate allele; HD = Huntington’s disease.
Clinical screening and definition of age at onset
Of the 220 individuals included, 185 (84%) were initially evaluated in the Department of Neurology and 35 (16%) in the Department of Psychiatry. Although initial evaluations were clinic-specific, most individuals were subsequently followed up through multidisciplinary HD care. All assessments were conducted directly by or under the supervision of senior clinicians with demonstrated experience in HD (PKP, RY, NK, SJ).
AAO was defined as the age at which the first clinically morbid symptom attributable to HD emerged – motor, psychiatric or cognitive. While motor onset (typically chorea) was most common, four individuals presented with psychiatric symptoms alone. Only individuals with manifest symptoms were included in the AAO-based analysis. Those with UHDRS-TMS DCL = 0 were considered presymptomatic and excluded.
Family history and genetic evaluation
Family history data were primarily obtained in the GCAT (Genetic Counseling and Testing) clinic, led by NR, through structured pedigree interviews and medical record review. A case was considered family history negative (FHN) if no first- or second-degree relatives were reported to have had chorea, progressive cognitive decline or a formal diagnosis of HD. Supplementary telephonic follow-ups were used to verify history and gather details on age at death, comorbidities or psychiatric symptoms in deceased or unavailable relatives.
Genomic DNA was extracted, and fluorescent PCR-based CAG repeat sizing was performed. In three of the suspected de novo expansion cases with available first-degree relatives, inheritance was confirmed using short tandem repeat (STR) marker analysis (GenePrint 10, Promega). These relatives were clinically asymptomatic and confirmed to have UHDRS-TMS DCL = 0.
To preserve participant anonymity, all age data are presented in age-range bins (e.g., 60–70 years) in both the manuscript and supplementary material. Exact values were recorded internally for clinical verification. Most families continue to be under longitudinal follow-up by NR through clinical care and ongoing community-based outreach.
Subgroup analysis and statistics
Subgroup analyses were conducted for juvenile-onset cases (AAO < 21 years), FHN cases and family-based correlations (parent–child and sibling–sibling pairs). Descriptive statistics, correlation analyses and group comparisons were performed.
Results
Demographics, AAO and CAG repeat length
The mean AAO of these subjects (N 220) was 39.2 ± 12.7 (range 4–72) years. Most individuals were from southern and eastern India, but several were from other parts of India (Supplementary Figure 1). The genders were almost equally represented, with a slight male preponderance (F 44%; M 56%). Mean upper and lower allele repeat lengths were 45.6 ± 8.3 repeats (range 38–113) and 17.9 ± 2.8 repeats (range 9–37), respectively (Figure 2A; Table S1, S2). The AAO showed a strong negative correlation with the longer allele (r = −0.69, p < 0.001) (Figure 2B). There were 26 families with more than 1 affected individual in this cohort.
(A) Distribution of longer and shorter alleles in Huntington’s disease patients (n = 220). (B–D) Correlation between CAG repeat length and age at onset (AAO) in (B) the entire cohort, (C) family history–negative cases and (D) juvenile–onset HD cases.
Reduced penetrance alleles (RPA), intermediate alleles (IA) and biallelic expansions
In this cohort, we found 12 patients with RPAs, and 7 of these had behavioral onset with a psychiatric syndrome as the primary diagnosis. Half of these cases (6/12) did not have a family history of chorea. We also found eight individuals with IAs (27–35) in our cohort. In two instances, an intermediate allele accompanied an FPA. In another three instances, the IAs were detected as longer alleles in the asymptomatic parents or siblings of HD patients with a de novo expansion (Supplementary Table 6). In addition, two young people (20–30 years) were detected to have IAs, who showed HD-like symptoms and in whom alternative diagnoses like acanthocytosis, Wilson’s disease and metabolic causes had been ruled out. Both showed evidence of caudate atrophy on MRI. Interestingly, a healthy parent of one of them also had an IA, an unexpected finding.
We also detected three cases of biallelic expansions (28/43, 27/47, 37/41), but without consanguineous parentage. In one biallelic individual (37/41), there was no history suggestive of HD either, as one parent died prematurely (4th decade) of unknown cause, and the other parent was alive and healthy (in 6th decade). These biallelic cases were clinically indistinguishable from the rest.
Juvenile-onset HD and large expansion alleles
Fourteen (8 F) individuals of the cohort had juvenile onset HD (JHD), having developed symptoms at quite a young age of 12 ± 4.5 years. Mean CAG repeat length was 72.1 ± 14.2. All of them, except one, were paternally transmitted. We found two JHD sibling pairs in this cohort. The highest CAG length observed was 113. The correlation between repeat number and AAO was more pronounced than in adult patients (r = −0.85, P < 0.001) (Figure 2D).
Inheritance pattern and family history negative (FHN) cases
Most patients (170 out of 220) had at least one parent affected with HD, with paternal inheritance (95, 55%) being slightly more common. The mean repeat length and dispersion of the expanded allele were higher in those with paternal transmission as compared to those with maternal transmission (48.1 ± 11.5 vs 44.7 ± 4.3, p = 0.01). However, there was no difference in AAO between the groups (36.2 ± 14.6 vs 38.9 ± 9.9 years, p = 0.19).
Of the remaining 50, family history was unavailable in 13 cases as they were orphaned or adopted at a young age. In the remaining 37 of them, neither of the parents nor parents’ siblings were known to have HD. We refer to them as FHN cases. In 26 FHN cases, both parents had lived at least 60 years of life with no symptoms, while in the remaining 11 FHN cases, 1 parent had died in middle age but had no symptoms to suggest HD, and the surviving parent (>60) too had no symptoms. However, in six instances, one parent had late-onset (6th–8th decade) behavioral changes, but no motor symptoms. There were two cases with HD symptoms in siblings, but both parents lived past 60 years without showing HD symptoms (Supplementary Table 3). In FHN cases, the mean size of the expansion was lower (42 repeats), and the AAO was higher (46 years), but the correlation between repeat number and AAO (r = 0.66, p < 0.001) was similar when compared to those who had an identifiable family history (Figure 2C).
We could test family members in three FHN cases where parents/siblings were available and consented for testing after the genetic counseling. In two of them, IAs (32 and 30 repeats) from asymptomatic parents had progressed to de novo mutations (43 and 44 repeats) in the offspring. In the third case with 48 repeats, the sibling had 32 repeats, while the living parent had normal alleles. The other parent had died in the 7th decade but never had any symptoms of HD. We suspect that this parent might have had an IA, which was stably transmitted in the case of the sibling but expanded in the patient. We also note that the sibling with IA had generalized anxiety. STR genotyping confirmed paternity and sibship in the three families described. Overall, a significant proportion (16%) of the cases in this cohort had apparently novel expansions.
CAG repeat expansion trends within a few families
From 26 families, we could identify 23 sibling pairs and 13 parent–offspring pairs (excluding de novo expansion pairs) with expanded repeats, of which 4 individuals were presymptomatic. Among sibling pairs, there were 15 paternal, 7 maternal and 1 unknown transmission (foster parents) (Supplementary Table 4). Repeat length of the expanded alleles and the AAOs strongly correlated between affected siblings (r = 0.95, 0.85, respectively), with a lesser mean AAO of 4.5 years in the younger sibling than the elder sibling (Figure 3A and 3B).
(A) Correlation plot of CAGs of sibling pairs. (B) Correlation plot of ages at onset (AAOs) of sibling pairs. (C) Correlation plot of CAGs of parent–offspring pairs. (D) Correlation plot of AAOs of parent–offspring pairs.
Among the 13 parent–offspring pairs (including 5 JHDs), the expanded allele had increased by about 14 repeats from parent (44.6 ± 3.1) to offspring (58.15 ± 20.4). Eight were paternal transmissions, while five were maternal. In nine pairs, there was an expansion in repeats (range 1–70); two pairs did not show any expansion, while two pairs showed contractions of one or two repeats from parent to offspring (Supplementary Table 5). The mean AAO of the parent and the offspring were 45.6 ± 14.6 (30 – 72) and 22.7 ± 9.2 (11 – 35) years, respectively, with a mean difference of 23 years (7–42 years). CAG repeats and AAOs of parents correlated strongly with those of offspring (r = 0.81, p = 0.001; r = 0.70, p = 0.01), respectively (Figure 3c and 3d).
Discussion
Despite several studies of HD in India, the lack of data regarding transmission and distribution of expanded alleles within families, as well as the occurrence of de novo expansion in the population, was the motivation for this study. We found strong evidence for CAG instability, although to varying extents, in this small set of families, and the prevalence of a range of alleles from intermediate alleles to very large expansions. Incidentally, we found intermediate alleles in young individuals suspected to have HD without any family history of HD. We present the insights derived from these findings to inform epidemiological research and clinical diagnosis.
Cohort characteristics and trends in families
The overall distribution of ages at onset, CAG repeat sizes and their correlation was broadly identical with those reported earlier from IndiaReference Chheda, Chanekar and Salunkhe12–Reference Moily, Kota and Anjanappa14 and from elsewhere.Reference Andrew, Paul Goldberg and Kremer3,Reference Li, Li and Dong15 The variations discovered in this well-documented cohort and their familial and genetic characteristics promote a more nuanced understanding of HD in South Asia.
Interestingly, two individuals with atypical clinical syndromes and symptoms of chorea had repeat sizes in the IA range. Whether IAs may be clinically innocuous or contribute to risk in some ways is thus still a matter for investigation.Reference Cubo, Ramos-Arroyo and Martinez-Horta16,Reference Ha and Jankovic17 Overall, out of five cases of IA transmission from four parents, two of them were stably transmitted, whereas three of them expanded to a pathogenic range in the offspring. Variable instability of IAs has been reported earlier.Reference Goldberg, McMurray and Zeisier18 Somatic instability in symptomatic IA carriers revealed that IAs expand somatically in a CAG and age-dependent manner, but whether this expansion or other genetic predispositions lead to disease is still unclear.Reference Ruiz De Sabando, Ciosi and Galbete19
Correlation of repeat sizes between siblings was stronger than in parent–offspring pairs. This suggests that the major expansion event perhaps occurs prior to gametogenesis,Reference Andrew, Paul Goldberg and Kremer3 whereas marked variation within some sibling pairs could be due to downstream events. In two-thirds of sibling pairs, younger siblings had an earlier onset, suggesting that the parental age at transmission influences anticipation. Although anecdotal, marked variation of AAO in siblings with similar repeats suggests a significant role of disease-modifying genetic variant in these cases. As expected, anticipation was high with paternal transmission, the most prominent one was seen in a JHD case (AAO-4 years, CAG-113) whose parent (CAG-43) was asymptomatic.Reference Ratna, Pasupulati and Nadella20 Such extremely large expansions have been observed in JHD, as when a child had more than 200 repeats, whereas the father had 43.Reference Nicolas, Devys and Goldenberg21 Whether these are a part of an inherent bias in chromatin organization or chromosomal arrangements or other genetic and/or environmental modifiers needs to be explored.
This study presents the first family-based, clinically annotated analysis of HD inheritance and allele diversity from India, with implications for genetic counseling and population-level risk estimates. The presence of intermediate and reduced penetrance alleles, apparent de novo mutations and cases without family history highlights the need for expanded clinical suspicion and molecular testing strategies, particularly in underdiagnosed regions. These findings can inform both epidemiological modeling and the design of future longitudinal genetic studies.
FHN cases, atypical alleles and de novo expansions – epidemiological implications
In almost a sixth of the patient sample, there was no obvious family history of an affected relative, but in a few cases, one of the parents developed late-onset behavioral symptoms, perhaps a manifestation of RPAs. Half (6/12) of the patients with RPAs had a spectrum of psychiatric symptoms, with mild or no motor symptoms. Such association of modestly expanded alleles with psychiatric symptoms has been reported.Reference Ellis, Tee and McAllister22 In addition, asymptomatic parents or siblings were detected to have an IA, while the index patient had an expanded allele, as has also been reported earlier.Reference Myers, MacDonald and Koroshetz10,Reference McNeil, Novelletto and Srinidhi11 Such prevalence of IAs in the family members of FHN cases poses a risk for de novo expansions in their offspring. The prevalence of “sporadic” HD differs across populations,Reference Almqvist, Elterman and MacLeod23,Reference Ramos-Arroyo24 owing to the varying incidence of de novo expansions of the IAs reported earlier (0%–14%).Reference Semaka, Collins and Hayden25,Reference Brocklebank, Gayán and Andresen26 The range of CAG repeats (39–50) and mean AAO of 46 in FHN cases of this sample suggests that clinicians should suspect HD in young individuals, even in the absence of family history.
The prevalence of IAs is reported to be about 6% in the general population and in 7% of those with HD.Reference Sequeiros, Ramos and Cerqueira27 The incidence of disease-causing expansions has been shown to correlate with the distribution of repeat sizes, the prevalence of IAs and at-risk haplotypes, thus influencing the overall HD prevalence rates in European, American, Asian and African populations.Reference Kay, Collins and Wright7 Exploring haplotypes of expanded alleles from FNH cases and in cases of other large expansion transmissions could shed light on the cis-acting factors contributing to expansion. In the 1000-genome data cohort, 7 healthy (presumably) individuals who had pathogenic expansions (36–52 repeats) were detected in 2486 healthy individuals.Reference Akçimen, Ross and Liao28 A study from Ecuador reported a high prevalence of IAs (18%) with significant ethnic differences,Reference Paz-y-Miño, Salazar-Ruales and García-Cárdenas29 while extremely short alleles as well as intermediate alleles were seen in a northern European non-HD population.Reference Sundblom, Niemelä and Ghazarian30 The occurrence of JHD alleles (6%) in this cohort is comparable with cohorts from elsewhere but much higher than other reports from India. A testing center from India reported the presence of 250 expanded alleles, including 9 RPAs, 7 large alleles > 60 repeats and 9 IAs (in negative samples) from 503 individuals who were tested, though clinical and family history details were not described.Reference Chheda, Chanekar and Salunkhe12 The epidemiology of intermediate and reduced penetrance alleles, along with their possible association with clinical symptoms in the population, thus needs investigation.
Biallelic expansions in three individuals in our sample could reflect the predisposition to allelic expansion in the general population, given the lack of consanguinity and at least one parent not having HD in all three cases. However, given the high rates of endogamy in southern India, the added risk for biallelic expansions and homozygosity in the subsequent generations of heterozygous patients’ needs to be noted. Significantly early age of marriage and reproduction (relative to AAO) in lower socioeconomic strata further increases this risk.
Limitations
Our study, based at a tertiary referral hospital, has an ascertainment bias and may lack sufficient statistical power to generalize the findings. However, as HD is a rare disorder, our data highlight the importance of family-based analysis to understand the transmission dynamics, rate of de novo expansions and the prevalence of late-onset psychiatric syndromes in such families.
Conclusions
Our study adds to the fund of existing knowledge of HD genetic epidemiology in India and brings some novel perspectives. Our findings suggest that novel expansions may not be rare, and there could be a load of expansion-prone alleles (IAs/RPAs) prevalent in the Indian population. This has significant epidemiological implications. Hence, there is an urgent need for both diagnosis-driven and population-based prevalence studies to estimate the current and projected burden of HD in India. Family-based genomic studies would help identify genetic modifiers of CAG instability, understand disease pathology and may even help develop personalized treatments.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/cjn.2025.10092.
Acknowledgments
HD patients were seen in the Movement Disorder Clinic and the Genetic Testing and Counseling Clinic at the National Institute of Mental Health and Neurosciences, Bengaluru, India. Material support for the work was provided by the Molecular Genetics Laboratory. Participants were included in the study after obtaining consent and institutional ethics clearance (Ref No. NIMH/DO/ETHICS SUB-COMMITTEE 29th MEETING/201).
Author contributions
NR: Conceptualization, methodology, formal analysis, original draft, review and editing.
SJ: Conceptualization, methodology, formal analysis, resources, review and editing.
MP: Methodology, formal analysis, resources, writing review and editing.
SP, SDV, GM: Investigation, data curation.
NK, RY, PKP: Investigation, writing review and editing.
BV, MV: Investigation, writing review and editing, supervision.
Funding statement
Dr Nikhil Ratna was supported by the Indian Council of Medical Research MD-PhD fellowship.
Competing interests
None of the authors has conflicts of interest to report.
