4.6. PROCEDIMENTOS E ANÁLISES MOLECULARES
4.6.4. Análises estatísticas
4.6.4.3. Procedimentos gerais
Os arquivos (.xls) contendo informações sobre a estrutura dos heredogramas (identificação da família, número de indivíduos, identificação da amostra, gênero, relações de parentesco, fenótipo) e com dados de genotipagem (marcador, alelos, identificação da amostra) foram organizados e formatados de acordo com o manual de instruções de cada programa e salvos em (.txt) como arquivos de entrada. Além disso, para a construção gráfica dos heredogramas e também dos haplótipos foi utilizado o programa HaploPainter (THIELE; NURNBERG, 2005).
As frequências alélicas, tanto para os marcadores SNP quanto para os microssatélites, da população brasileira envolvida no estudo foram estimadas pelo programa Merlin (ABECASIS et al., 2002) disponível na plataforma Helix. Os dados de frequência foram salvos em arquivo (.txt) e posteriormente formatados de acordo as necessidades de cada programa utilizado nas análises.
5. RESULTADOS
5.1. GRUPO AMOSTRAL
As amostras selecionadas estão apresentadas, e distribuídas conforme as tabelas a seguir: amostras de indivíduos (não relacionados) com gagueira desenvolvimental persistente familial e, casos controle, tabela 13; amostras de famílias completas com gagueira desenvolvimental persistente, tabela 14.
Tabela 13. Distribuição do número de indivíduos, não relacionados, com gagueira desenvolvimental
persistente familial e, casos controle.
Origem Identificação da amostra
Distribuição dos indivíduos fenótipo e gênero
TOTAL Gagos Fluentes
Masc. Fem. Masc. Fem.
CEES – Unesp
Marília BRMII 83 32 0 0 115
SAG – IBB – Unesp
Tabela 14. Distribuição do número de famílias com gagueira desenvolvimental persistente, não
relacionadas.
Origem N Identificação das famílias
Distribuição dos indivíduos fenótipo e gênero
TOTAL DE MEMBROS NA FAMÍLIA Gagos Fluentes
Masc. Fem. Masc. Fem.
CEES – Unesp Marilia 1 BR_01 2 0 0 3 5 2 BR_02 2 2 1 2 7 3 BR_03 3 0 0 1 4 4 BR_04 1 1 0 1 3 5 BR_05 2 0 0 1 3 6 BR_06 3 1 3 1 8 7 BR_07 4 0 0 1 5 8 BR_08 3 0 1 1 5 9 BR_09 4 0 1 1 6 10 BR_10 2 0 2 8 12 11 BR_11 1 2 6 2 11 12 BR_12 3 2 0 2 7 13 BR_13 5 1 0 2 8 14 BR_14 2 0 0 1 3 15 BR_15 2 1 4 0 7 16 BR_16 3 1 1 3 8 17 BR_17 3 1 2 2 8 18 BR_18 2 0 3 2 7 19 BR_19 1 1 4 6 12 20 BR_20 3 0 2 5 10 21 BR_29 2 0 2 2 6 22 BR_26 2 0 0 2 4 23 BR_30 3 0 7 8 18 24 BR_31 2 0 1 4 7 25 BR_33 1 2 1 0 4 26 BR_34 3 0 4 9 16 27 BR_47 4 1 2 5 12 28 BR_48 2 0 4 5 11 29 BR_49 3 0 3 2 8 30 BR_50 4 0 0 5 9 31 BR_51 2 1 2 4 9 FOFITO-USP São Paulo 32 BR_21 5 1 1 2 9 33 BR_22 2 1 0 1 4 34 BR_23 1 1 1 0 3 35 BR_24 2 2 1 1 6 36 BR_25 1 1 0 0 2 37 BR_27 2 1 0 1 4 38 BR_28 2 0 0 1 3 39 BR_35 1 1 1 4 7 40 BR_38 1 1 0 1 3 41 BR_45 2 0 1 2 5 42 BR_52 3 0 4 6 13 43 BR_53 2 2 3 3 10 TOTAL 43 famílias 103 28 68 113 312
A figura 5, 6, 7, 8, 9, 10, 11, 12 representam os heredogramas, em ordem crescente de identificação, referentes às 43 famílias estudadas. Todas, independentemente do número de indivíduos foram submetidas à genotipagem por SNPs e análise de ligação. Somente, duas famílias (BRPD47 e BRPD50) apresentaram sugestivos indicativos de ligação sob o padrão de herança dominante no cromossomo 10, quando combinadas (APÊNDICES E, F, G).
Figura 5. Heredogramas: BRPD01, BRPD02, BRPD03, BRPD04, BRPD05, BRPD06. BRPD01 BRPD03 BRPD04 BRPD05 BRPD06 69
Figura 6. Heredogramas: BRPD07, BRPD08, BRPD09, BRPD10, BRPD11, BRPD12.
BRPD07
BRPD11
BRPD12
Figura 7. Heredogramas: BRPD13, BRPD14, BRPD15, BRPD16, BRPD17, BRPD18.
BRPD14 BRPD15
BRPD17 BRPD18
Figura 8. Heredogramas: BRPD19, BRPD20, BRPD21, BRPD22, BRPD23, BRPD24, BRPD25, BRPD26. BRPD20 BRPD22 BRPD23 BRPD24 BRPD25 BRPD26 72
Figura 9. Heredogramas: BRPD27, BRPD28, BRPD29, BRPD30, BRPD31, BRPD33. BRPD27 BRPD28 BRPD30 BRPD31 BRPD33 ? 73
Figura 10. Heredogramas: BRPD34, BRPD35, BRPD38, BRPD45, BRPD47. BRPD38 BRPD45 BRPD47 74
Figura 11. Heredogramas: BRPD48, BRPD49, BRPD50
BRPD49
BRPD50
Figura 12. Heredogramas: BRPD51, BRPD52, BRPD53.
BRPD51
BRPD53
5.2. ARTIGOS CIENTÍFICOS
A partir dos dados obtidos pelas análises foram redigidos dois artigos científicos descritos a seguir (a formatação de cada artigo segue as instruções de cada revista).
CORRESPONDENCE
Evaluation of the association between polymorphisms
at the DRD2 locus and stuttering
Journal of Human Genetics advance online publication, 10 March 2011; doi:10.1038/jhg.2011.29
Based on the report of Lan et al.,1we sought to replicate the association between stuttering and single-nucleotide polymorphisms (SNPs) that reside in the dopamine D2 receptor (DRD2) gene in additional populations. The only individual significant association observed by Lan et al. was with the C allele of rs6277, a synonymous SNP (Pro319Pro) within the coding sequence of this gene. In their Han Chinese sample of 112 cases and 112 controls, this allele occurred at a fre- quency of 0.96 in cases and 0.88 in controls, with a reported P-value of 0.001.
We studied this polymorphism as well as another DRD2 synonymous SNP, rs6275, also studied by Lan et al., in two additional case– control populations that together represented a larger sample. The first was a group of 50 cases and 50 sex-matched controls of mixed ancestry2from Sao Paulo State, Brazil, while the second was a group of 214 cases and 451
neurologically normal controls of western European origin.3All subjects provided writ- ten informed consent under institutionally approved human subjects research protocols. Speech diagnosis was performed using the Stuttering Severity Instrument, 3rd Edition (SSI-3),4and as previously described.3Geno- types were determined by dye terminator sequencing of both strands using an ABI 3730 XL capillary instrument.
Our first analysis determined that the genotypes at rs6277 and rs6275 did not deviate significantly from Hardy–Weinberg equilibrium expectation in either Brazilian (P¼0.60, P¼0.62) or western European populations (P¼0.59, P¼0.43). The geno- typic distributions we observed are shown in Table 1. In these populations, no signifi- cant differences between cases and controls were observed for allele frequencies of rs6277 (P¼0.26 in the Brazilian population and
P¼0.25 in the western European population) or rs6275 (P¼0.77 and P¼0.13, respectively). Analysis of haplotypes containing these two SNPs failed to show any significant associa- tion with stuttering (P¼0.11–0.77).
Our results differ substantially from those of Lan et al., who reported that the C allele of rs6277 is more frequent in cases than in controls. However, because this allele exists at a frequency of 88% in normal controls, it seems unlikely to be a significant contributor to stuttering by itself in the Han Chinese population, where stuttering has been esti- mated to occur in only 1% of adults.5 In addition, Lan et al. reported a somewhat paradoxical finding regarding rs6277 diplo- types. While homozygosity for the C allele was more common in cases (92.9 vs 77.7% in controls), having one copy of the C allele was more common in controls (19.6 vs 7.1% in cases). This would seem to suggest a situation in which the C allele carries a slightly increased risk when present in two copies but has a large opposite effect when present in one copy.
It is possible that an authentic association with rs6277 exists in Han Chinese (who speak Mandarin, a hieroglyphic language) but not in Brazilians or western Europeans (who speak alphabetic languages). In both our western European cohort and our Brazilian cohort, the C allele of rs6277 is much less frequent than in the Han Chinese, where it is the most common allele. This difference may help obscure the true extent of asso- ciation more generally. However, our data do not support the conclusion that the C allele of rs6277 is associated with stuttering in general.
ACKNOWLEDGEMENTS
This work was supported by NIDCD Intramural Grant DC-000046-11 and by FAPESP #2007/02561-9
Table 1 Allelic association test for two SNPs in DRD2 with susceptibility of stuttering
Caucasian Chinese Brazilian
Controls (N¼451) Cases (N¼214) Controls (N¼112) Cases (N¼112) Controls (N¼50) Cases (N¼50) rs6275 (C/T) CC 234 99 17 19 19 20 CT 178 91 63 62 26 22 TT 39 24 32 31 5 8 T freq. 0.28 0.32 0.57 0.55 0.36 0.38 P value 0.13 0.78 0.77 rs6277 (T/C) TT 142 59 3 0 11 9 TC 214 104 22 8 28 24 CC 95 51 87 104 11 17 C freq. 0.45 0.48 0.88 0.96 0.50 0.58 P value 0.25 0.001 0.26
and #2010/00501-1. We thank the stuttering research subjects for their participation.
Changsoo Kang1, Bianca Santos Domingues2, Eduardo Sainz1, Carlos Eduardo Frige´rio Domingues2, Dennis Drayna1 and Danilo Moretti-Ferreira2 1National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD, USA
and2Department of Genetics, Bioscience Institute, Sao Paulo State University (Unesp), Botucatu, Brazil E-mail: drayna@nidcd.nih.gov
1 Lan, J., Song, M., Pan, C., Zhuang, G., Wang, Y., Ma, W. et al. Association between dopaminergic genes (SLC6A3 and DRD2) and stuttering among Han Chinese. J. Hum. Genet. 54, 457–460 (2009).
2 Lins, T., Vieira, R., Abreu, B., Grattapaglia, D. & Pereira, R. Genetic composition of Brazilian population samples based on a set of twenty eight ancestry informative SNPs. Am. J. Hum. Biol. 22, 187–192 (2010).
3 Kang, C., Riazuddin, S., Mundorff, J., Krasnewich, D., Friedman, P., Mullikin, J. et al. Mutations in the lysosomal enzyme-targeting pathway and persistent stuttering. N. Engl. J. Med. 362, 677–685 (2010). 4 Riley, G. D. Stuttering Severity Instrument for Children
and Adults 3rd edn (Pro-Ed, Austin, TX, 1994). 5 Bloodstein, O & Ratner, N. B. A Handbook on Stuttering
6th edn (Thomson Delmar Learning, Clifton Park, NY, 2008).
5.2.2. Artigo científico 02
A genetic linkage study in Brazil identifies a new locus for persistent developmental stuttering on chromosome 10
Carlos Eduardo Frigerio Domingues1,2, Cristiane Moço Canhetti de Olivera3, Breila Vilela de Oliveira 2, Fabiola Staróbole Juste4, Claudia Regina Furquim de Andrade4, Celia Maria Giacheti3, Danilo Moretti-Fereira2, Dennis Drayna1#,
1. National Institute on Deafness and Other Communication Disorders, National
Institutes of Health, Rockville, MD, USA
2. Department of Genetics, Biosciences Institute, Sao Paulo State University (Unesp),
Botucatu, SP, Brazil
3. Department of Speech Pathology, Education and Health Studies Center, Sao Paulo
State University (Unesp), Marilia, SP, Brazil
4. Department of Physiotherapy, Communication Science and Disorders, Occupational
Therapy, Faculty of Medicine, Sao Paulo University (FMUSP), Sao Paulo, SP, Brazil
# To whom correspondence should be addressed at: NIDCD/NIH
5 Research Court, Room 2B-46
Rockville, MD 20850 USA
Tel. 301-402-4930
e-mail: drayna@nidcd.nih.gov
Abstract
Although twin, adoption, and family studies demonstrate that genetic factors are
involved in the origins of stuttering, the mode of transmission of the disorder in families
is not well defined and stuttering is considered a genetically complex trait. We
performed a genome-wide linkage scan in a group of 43 Brazilian families, each
containing multiple cases of persistent developmental stuttering. Linkage analysis
under a dominant model of inheritance generated significant evidence of linkage in two
Brazilian families, with a combined maximum single point LOD score of 4.02 and a
multipoint LOD score of 4.28 on chromosome 10q21. This demonstrates the presence
of a novel variant gene at this locus that predisposes individuals to stuttering, which
provides an opportunity to identify novel genetic mechanisms that underlie this
1. Introduction
Although the etiology of developmental stuttering is not well understood, there
is a consensus that genetic factors are involved, and studies of twins (Howie, 1981;
Andrews et al., 1991; Felsenfeld et al., 2000; Ooki, 2005; Dworzynski et al., 2007;
Fagnani et al., 2011; Rautakoski et al., 2012)and families (Kidd et al., 1978; Seider et
al., 1983; Cox et al., 1984; Viswanath et al., 2004) have contributed to the substantial
evidence for the role of inherited factors in the etiology of this disorder. Despite the
extensive evidence for genetic factors, however, the exact mode of transmission in
families is not well defined, and it is unlikely to be the same in all families and
populations (Alm et al., 2007). For example, a number of linkage loci have been
identified in consanguineous families, particularly from Pakistan (Riaz et al., 2005;
Raza et al., 2010; Raza et al., 2012), while studies in outbred families in the United
States have not demonstrated linkage at these loci (Shugart et al., 2004; Suresh et al.,
2006). The goal of this study was to identify genetic factors underlying persistent
developmental stuttering in the Brazilian population, which displays a high degree of
genetic admixture, and is thus quite different from the populations used for previous
genetic studies of stuttering. To do this, we have performed a genome-wide linkage
scan in a group of Brazilian families, each containing multiple cases of persistent
developmental stuttering.
2. Materials and Methods
The study was approved by the respective institutional IRB’s (Research Ethics
Committee of Botucatu Medical School, UNESP/Brazil n.3441/2010-CEP; National
Committee for Ethics Research, n.16349; National Institutes of Health, protocol 97-DC-
subject, was obtained from all participants. Blood and recorded speech samples were
collected from all available family members. A total of 43 unrelated Brazilian families
were enrolled from Sao Paulo State, Brazil. The sample consisted of 233 individual
participants, 130 of whom were diagnosed as affected with persistent developmental
stuttering.
Stuttering diagnosis was determined by at least two speech pathologists and was
established based on self-expressive speech samples (Andrade et al., 2004) of at least
200 syllables elicited by visual stimuli (pictures) adapted for different age groups.
Affected individuals were defined as those with a stuttering dysfluency rate of 3% and
a score of 11 points or more from the Stuttering Severity Instrument, 3rd Edition (SSI-
3) (Riley, 1994), were age 6 years and older, had stuttered for at least 6 months, and had
at least one additional affected family member. Exclusion criteria were: family history
of genetic or non-genetic neurological disorders (e.g., dystonia, extra pyramidal disease,
mental disorder, epilepsy, attention deficit hyperactivity disorder (ADHD), psychiatric
symptoms or conditions, oral communication impairment not compatible with age,
conductive or neurosensory hearing loss, or other developmental conditions) that could
generate errors in diagnosis.
Genotyping was performed using the Illumina HumanLinkage-24 BeadChip that
assayed 5913 single nucleotide polymorphisms (SNPs) in all 233 individuals. Samples
from twenty-six Brazilian unaffected individuals were genotyped to estimate SNP allele
frequencies in the Brazilian population. The quality of SNP genotypes was evaluated
according to the manufacturer’s protocol using Illumina®GenomeStudio V2009.2 software (analysis parameters: cluster separation, call frequency, AB R mean, AB T
mean, Mendelian inheritance, heterozygote excess, minor allele frequencies; available
genotyping_data_analysis.pdf). Nine percent of SNPs failed to meet the recommended
criteria and were excluded, leaving a total of 5379 SNPs analyzed for linkage.
Single-marker parametric linkage analysis was performed using SuperLink v1.6
(Hoffmann and Lindner, 2005) from the easyLINKAGE-Plus v5.08 package (available
at: http://sourceforge.net/projects/easylinkage/) for SNP data. Confirmation of results
from SNPs was done by genotyping microsatellite markers on chromosome 10 with
standard fluorescent methods using an ABI 3730 XL capillary instrument and
GeneMapper ® v.4.0 (Applied Biosystems, USA), followed by MLINK analysis (Cottingham et al., 1993)and SUPERLINK online V1.5 (Fishelson and Geiger, 2002;
Silberstein et al., 2006). SLINK was used to calculate simulated LOD scores using a
single-marker power calculation with 1,000 replicates (Ott, 1989). Microsatellite allele
frequencies were estimated by genotyping 48 Brazilian normal control individuals.
MERLIN (available at: http://www.sph.umich.edu/csg/abecasis/Merlin/download/) was
used to calculate most likely haplotypes(Abecasis et al., 2002) and HAPLOPAINTER
(available at: http://sourceforge.net/projects/haplopainter/) was used to plot the pedigree
with the haplotypes (Thiele et al., 2005). Analysis was initially performed on both
individual families and on the combined family set with the disease allele frequency set at
0.01.
3. Results
Analysis of the combined family set of 43 families under dominant, recessive,
and additive models of inheritance failed to generate significant evidence of linkage.
Analysis of individual families demonstrated suggestive evidence of linkage in two
families (BRPD47 and BRPD50), while all other individual families displayed no
evidence for linkage under any model of inheritance. Families BRPD47 and BRPD50
(58.81 Mb) on chromosome 10q. These markers generated LOD scores of 1.59 and
1.32 respectively under a dominant model of inheritance, which was viewed as the most
plausible model based on the segregation of stuttering in these two families.
Subsequent analysis under recessive and additive models gave reduced scores in these
families. Analysis of both families together under a dominant model (penetrance classes
0.01, 0.99, 0.99) produced positive LOD scores at multiple markers, including
rs1268722 (LOD=3.11); rs77839 (LOD=2.05); rs1917810 (LOD=1.97); rs913034
(LOD=1.84) and rs1427209 (LOD=1.83).
Based on these results, we performed fine mapping using microsatellite markers
that extended from markers D10S34 (48.38 Mb) to D10S535 (76.34Mb) (Genome build
36/hg18). Analyses of these markers under a dominant model produced a maximum
single-marker LOD score of 2.57 in BRPD47 at D10S1643, while the maximum score
of 1.74 in BRPD50 was generated at multiple markers in this region, extending from
D10S604 (44.84 Mb) to D10S2480 (70.32 Mb). The analysis in the two families
combined generated a pairwise LOD score of 4.02 at marker D10S220 (Table 1B).
Multi-point analysis based on six microsatellite markers using the same inheritance
model generated higher LOD scores. For BRPD47 a LOD score of 2.49 was obtained at
D10S1790 (55.2 Mb) and the LOD score exceeded 2.4 across the region from D10S604
(44.84Mb) through D10S529 (71.83Mb). In BRPD50, a maximum score of 1.78 was
generated, with a flat LOD score curve between D10S604 (44.84 Mb) and D10S1670
(68.87 Mb). Analysis of these two families combined generated a maximum LOD score
of 4.28 at marker D10S1790 (Table 1C).
The observed linkage scores were compared to the hypothetical maximum
single-marker scores obtained from these two families using simulation, assuming that
observed scores can exceed the simulated scores). SLINK (Ott, 1989) generated
maximum simulated LOD scores of 2.58 and 1.74 for BRPD47 and BRPD50
respectively, and a score of 4.32 for the families combined. These are close to the
maximum linkage scores we observed, suggesting that our linkage results explain all of
the observed segregation of stuttering in these two families.
The haplotypes observed across the 20 microsatellite markers that showed
evidence of linkage are presented in Figure 1. In family BRPD47, the haplotypes for
individual 47_20 were inferred, as this individual was not available for study.
Designation of this individual as affected is based on family report, which if correct,
indicates that this individual stuttered without carrying the haplotype associated with
stuttering in other family members. The haplotypes that segregate with stuttering differ
in these two families. Although our haplotype results are consistent with our linkage
findings in this region in both families, they suggest that these families do not share a
mutation of common origin at this locus.
4. Discussion
While persistent stuttering has failed to demonstrate a clear mode of inheritance
in families in a number of previous studies, transmission of the disorder is consistent
with autosomal dominant, perhaps with reduced penetrance, in two Brazilian families in
our study. Linkage analysis under this model of inheritance generated highly significant
evidence for linkage on chromosome 10q, with a combined multipoint LOD score of
4.28. Although our evidence for linkage came from only two of the 43 families in our
sample, we note that the majority of the remaining families were small and presented
low power to detect linkage individually. Our observation of linkage in a subset of
families is also consistent with previous results that have demonstrated a high degree of
2005; Suresh et al., 2006; Raza et al., 2010; Kang et al., 2010; Raza et al., 2012). Thus,
it is not surprising that our study found evidence of linkage in only a subset of the
families, and that the linkage locus we identified in the Brazilian population has not
been previously observed for stuttering.
One individual (individual 47_20 in family BRPD47) was designated as affected
by family report and this individual’s haplotypes were inferred, as he was not available
for this study. If the diagnosis and haplotypes for this individual are correct, he
represents a phenocopy because he stuttered but did not carry the disease-associated
haplotype in this family. Such phenocopies have been observed in previous studies of
large stuttering families (Kang et al., 2010). Such individuals indicate that even when a
significant linkage score can be obtained under a Mendelian model of inheritance,
stuttering can display non-Mendelian features.
The Brazilian population is characterized by a high degree of admixture.
Genetic studies in such populations can use different genotypes and haplotypes that
derive from the different racial or ethnic ancestral groups to improve the power to
identify and localize disease causing alleles. For this reason the Brazilian population is
considered a valuable resource for mapping complex disease traits (Giolo et al., 2012).
Previous studies in other populations, especially those containing consanguineous
families have typically produced strongest evidence for linkage under a recessive
model. In contrast, the families in our sample have no evidence for consanguinity and
demonstrate linkage under a dominant model of inheritance, with evidence for different
mutations at the same locus. This suggests that these families displaying linkage to
chromosome 10q provide an opportunity to identify novel genetic mechanisms that
5. Conclusion
Our genome-wide linkage scan in the Brazilian population identified linkage in
two families to chromosome 10q21 under dominant inheritance, which represents a
novel locus and a novel mode of inheritance for this disorder.
Acknowledgements
We thank M. Hashim Raza, Eduardo Sainz, Alejandro Schaffer, Thomas
Friedman and Andrew Griffith for technical guidance and comments on the manuscript,
and the family members who participated in this research study. This work was
supported by the National Institute on Deafness and Other Communication