Caria, H

Caroça, C

Fialho, G

Morim de Matos, T

Paço, J

English

Repositório Comum

DFNB1	  locus	  analysis	  in	  São	  Tomé	  and	  Príncipe	  popula;on	  	  Cris&na	  Caroça1,2,3;	  Tiago	  Morim	  de	  Matos3,4;	  Graça	  Fialho4;	  João	  Paço1,2;	  Helena	  Caria3,4	  	  1University	  Center	  of	  Otorhinolaryngology	  -­‐	  Hospital	  CUF	  Infante	  Santo,	  Lisbon,	  Portugal	  	  2Nova	  Medical	  School	  –	  Faculty	  of	  Medical	  Sciences	  of	  Lisbon,	  Lisbon,	  Portugal	  3Higher	  School	  of	  Health,	  Polytechnic	  Ins&tute	  of	  Setúbal,	  Setúbal,	  Portugal	  4Biosystems	  &	  Integra&ve 	  Sciences	  Ins&tute	  ( BioISI),	  Faculty	  of	  Sciences	  of	  the	  University	  of	  Lisbon,	  Lisbon,	  Portugal	  	  Deafness	  is	  one	  of	  the	  leading	  causes	  of	  disability	  in	  the	  world.	  The	  prevalence	  of	  hearing	  loss	  is	  highest	  in	  South	  Asia	  and	  sub-­‐Saharan	  Africa1.	  This	  condi&on	  could	  be	  caused	  by	  environmental	  condi&ons,	   gene&cs,	   aging,	   or	   a	   combina&on	  of	   these	   factors2.	   Congenital	   deafness	  occurs	   in	  approximately	   1	   in	   1,000	   live	   births;	   while	   50%	   of	   these	   cases	   are	   hereditary	   in	   developed	  countries,	   the	   contribu&on	   of	   gene&c	   muta&ons	   to	   deafness	   decreases	   in	   underdeveloped	  countries	  due	  to	  the	  higher	  relevance	  of	  environmental	  causes.	  GJB2	   (connexin	  26)	  muta&ons	  have	   been	   iden&fied	   as	   the	   most	   common	   cause	   of	   hereditary	   hearing	   loss	   in	   many	  popula&ons3.	  Two	  large	  GJB6	  dele&ons,	  del(GJB6-­‐D13S1830)	  and	  del	  (GJB6-­‐D13S1854),	  are	  also	  a	  significant	  cause	  of	  HL	  in	  several	  countries.	  These	  two	  genes	  comprise	  the	  DFNB1	  locus,	  which	  has	  sparsely	  been	  inves&gated	  in	  African	  HL	  pa&ents.	  	  In	   this	   study	  we	  aimed	  at	   es&ma&ng	   	   the	  prevalence	  of	  GJB2	   coding	  muta&ons	   and	   the	   two	  large	  GJB6	   dele&ons	   in	   a	   sample	   of	   São	   Tomé	   and	   Príncipe,	   	   It	   is	   the	   first	   study	   of	   this	   kind	  carried	  out	  in	  this	  popula&on.	  Sequencing	  results	  allowed	  for	  the	   iden&fica&on	  of	  coding	  variants	  (table	  1)	  and	  also	  for	  the	  determina&on	  of	  genotypic	  frequencies	  of	  ten	  noncoding	  variants	  (table	  2).	  	  	  Some	  pathogenic	  and	   likely	  pathogenic	  GJB2	   coding	  muta;ons	  have	  been	   iden;fied	   in	  this	  study:	  	  Bilateral	  HL	  (2/132	  alleles)	  •  p.Met34Thr	  -­‐	  pathogenic	  variant	  •  p.Lys112Met	  -­‐	  likely	  pathogenic	  variant	  (based	  in	  in	  silico	  predic&on	  at	  hdp://deafnessvaria&ondatabase.org/)	  	  Unilateral	  HL	  (1/70	  alleles)	  •  p.Val167Met	  -­‐	  likely	  pathogenic	  variant	  (based	  in	  silico	  predic&on	  at	  the	  same	  website)	  	  Controls	  (2/296	  alleles)	  •  p.Val37Ile	  -­‐	  pathogenic	  variant	  •  p.Val167Met	  -­‐	  likely	  pathogenic	  variant	  	  Two	  synonymous	  changes	  p.Arg75=	  and	  p.Asn62=	  have	  been	  iden&fied	  in	  HL	  pa&ents	  and/or	  controls.	  	  The	  non-­‐sub-­‐Saharan	  gene&c	  influence	  in	  São	  Tomé	  and	  Príncipe	  popula&on,	  most	  likely	  due	  to	  admixture	  with	  Portuguese,	  was	  adested	  by	  the	  iden&fica&on	  of	  coding	  muta&ons	  commonly	  found	  in	  Europe	  and	  Asia:	  p.Met34Thr,	  p.Val37Ile,	  p.Arg127His	  and	  p.Val153Ile.	  	  Interes&ngly,	   the	   three	   groups	   of	   subjects	   and	   the	   genotype	   concerning	   the	   c.*84T>C	  variant	  are	  not	   independent,	  as	  shown	   in	   table	  2	   (Chi-­‐square	  test,	  p=0.036).	   Indeed,	   the	  allele	   c.*84C	   is	  overrepresented	   in	   the	  bilateral	  HL	  pa&ents	   compared	  with	   the	   controls	  (Chi-­‐square	  test,	  p=0.011).	  Our	  study	  suggests	  that	  the	  contribu&on	  of	  GJB2	  coding	  muta&ons	  to	  HL	  in	  São	  Tomé	  and	  Príncipe	   is	   small,	   similarly	   to	   what	   has	   been	   observed	   in	   other	   studies	   of	   sub-­‐Saharan	  popula&ons2.	  This	  is	  so	  despite	  the	  admixture	  to	  some	  degree	  with	  European	  popula&ons,	  namely	  the	  Portuguese4.	  None	  of	  the	  GJB6	  dele&ons	  was	  iden&fied	  in	  the	  91	  bilateral	  HL	  pa&ents,	  44	  unilateral	  HL	  pa&ents	  or	  179	  controls	  analyzed.	  Subjects	  •  A	  total	  of	  316	  individuals	  (136	  HL	  pa&ents	  and	  180	  controls),	  ranging	  from	  2	  to	  35	  years	  old,	  agreed	  to	  par&cipate	  in	  this	  study.	  The	  subjects	  were	  recruited	  during	  consulta&on	  provided	  by	  the	  humanitarian	  missions	  in	  São	  Tomé	  and	  Príncipe,	  at	  hospitals,	  schools	  and	  an	  hotel,	  over	  a	  period	  from	  February	  2012	  to	  May	  2014.	  All	  pa&ents	  and	  controls	  answered	  a	  clinical	  ques&onnaire	  iden&fying	  risk	  factors	  (family	  history	   of	   HL,	   consanguinity,	   malaria	   infec&on,	   pre-­‐natal	   and	   perinatal	   history,	   and	   other	   infec&ons	  history),	  clinical	  history	  and	  otolaryngology	  observa&on.	  Audiological	  examina;on	  •  All	   316	   individuals	   were	   evaluated	   regarding	   their	   hearing	   status	  with	   Pure	   Tone	   Audiogram	   (PTA)	   -­‐	  	  Madsen	  Midimate	  622	   	  or	  Auditory	  Brainstem	  Response	   (ABR)	   -­‐	  Vivosonic	   Integrity	  V500	  audiometer	  	  depending	   on	   collabora&on.	   The	   audiometric	   exams	  were	   carried	   out	  without	   an	   audiometric	   cabin,	  with	  earphones-­‐TDH39,	   in	  a	  closed	  room,	  with	  a	   level	  of	  noise	  measured	  by	   iPhone	  de	  SchabelDoesIT	  GbR,	  Munich,	  Germany	  (version	  1.0.0),	  considered	  acceptable,	  based	  on	  ANSI	  S3.1-­‐1999	  (R2013).	  	  DFNB1	  analysis	  •  Peripheral	   blood	   	   samples	  were	   collected	   in	  Guthrie	   paper	   cards	   arer	   informed	   consent	   being	  signed.	   Approval	   of	   the	  Medical	   Ethics	   Commidee	  was	   previously	   obtained.	   Genomic	   DNA	  was	  obtained	   from	   each	   blood	   sample	   using	   a	   commercially	   available	   kit	   (QIAamp®	   DNA	  micro	   kit,	  Qiagen)	  according	  to	  the	  manufacturer’s	  instruc&ons.	  All	  DNA	  samples	  were	  stored	  at	  –20	  °C	  un&l	  analysis.	  Of	  the	  316	  subjects,	  sequencing	  of	  the	  coding	  region	  of	  the	  GJB2	  gene	  was	  performed	  in	  101	  pa&ents	  (66	  bilateral	  +	  35	  unilateral)	  and	  148	  controls.	  These	  249	  subjects,	  as	  well	  as	  other	  25	  bilateral	  HL	  pa&ents,	  9	  unilateral	  HL	  pa&ents	  and	  31	  controls,	  were	  tested	  for	  both	  GJB6	  dele&ons.	  	  •  The	  GJB2	  coding	  region	  was	  amplified	  in	  a	  single	  fragment	  of	  928	  bp,	  as	  previously	  described5.	  The	  PCR	  products	  were	  purified	  with	  the	  JetQuick	  PCR	  Purifica&on	  Spin	  Columns	  kit,	  and	  subsequently	  sequenced	   by	   an	   outsource	   company.	   The	   presence	   of	   the	  GJB6	   dele&ons	   was	   inves&gated	   by	  mul&plex	  PCR,	  as	  described	  by	  del	  Cas&llo	  et	  al	  20056.	  Ø The	  role	  of	  GJB2	  coding	  muta&ons	  in	  HL	  in	  São	  Tomé	  and	  Príncipe	  seems	  to	  be	  of	  lidle	  significance.	  	  Ø Our	   study,	   however,	   shows	   the	   existence	   of	   pathogenic	   and	   likely	   pathogenic	  coding	  variants	  in	  São	  Tomé	  and	  Príncipe	  popula&on.	  	  Ø Thus,	   although	   no	   bi-­‐allelic	   HL	   pa&ents	   have	   been	   iden&fied	   in	   our	   sample,	   the	  occurrence	  of	  GJB2-­‐related	  HL	  in	  this	  popula&on	  is	  possible.	  The	  prevalence	  of	  either	  the	   two	   large	  GJB6	  dele&ons,	   if	  present,	   is	  expected	   to	  be	   less	   than	  1/91	   (1,1%)	   in	  bilateral	  HL	  pa&ents	  from	  São	  Tomé	  and	  Príncipe.	  	  GJB2	  variants	   Bilateral	  HL	   Unilateral	  HL	   Controls	  c.101T>C	  (p.Met34Thr)	   1/66	   0/35	   0/148	  c.109G>A	  (p.Val37Ile)	   0/66	   0/35	   1/148	  c.186C>T	  (p.Asn62=)	   0/66	   0/35	   1/148	  c.225G>T	  (p.Arg75=)	   1/66	   0/35	   4/148	  c.335A>T	  (p.Lys112Met)	   1/66	   0/35	   0/148	  c.380G>A	  (p.Arg127His)	  	   0/66	   0/35	   1/148	  c.457G>A	  (p.Val153Ile)	   0/66	   0/35	   1/148	  c.499G>A	  (p.Val167Met)	   0/66	   1/35	   1/148	  GJB2	  noncoding	  variant	  	  Genotypes	  	   Bilateral	  HL	   Unilateral	  HL	   Controls	   P-­‐Value	  (Chi-­‐Square	  test)	  c.-­‐22-­‐12C>T	  	   CC	   13/22	   5/9	   14/21	   0.651*	  CT	   6/22	   4/9	   6/21	  TT	   3/22	   0/9	   1/21	  c.-­‐15C>T	  	   CC	   51/61	   28/34	   124/142	   0.487*	  CT	   9/61	   6/34	   18/142	  TT	   1/61	   0/34	   0/142	  c.-­‐14G>A	  	   GG	   0/61	   0/34	   0/143	   NA	  GA	   0/61	   0/34	   1/143	  AA	   0/61	   0/34	   0/143	  c.-­‐7G>A	  	   GG	   64/64	   35/35	   147/148	   NA	  GA	   0/64	   0/35	   1/148	  AA	   0/64	   0/35	   0/148	  c.-­‐6T>A	  	   TT	   66/66	   35/35	   144/148	   0.277*	  TA	   0/66	   0/35	   4/148	  AA	   0/66	   0/35	   0/148	  c.*78A>T	  	   AA	   56/58	   32/32	   126/126	   NA	  AT	   2/58	   0/32	   0/126	  TT	   0/58	   0/32	   0/126	  c.*84T>C	  	   TT	   10/57	   9/32	   34/122	   0.036	  TC	   19/57	   15/32	   56/122	  CC	   28/57	   8/32	   32/122	  c.*96A>G	  	   AA	   0/35	   0/21	   0/96	   NA	  AG	   0/35	   0/21	   1/96	  GG	   0/35	   0/21	   0/96	  c.*104A>T	  	   AA	   18/33	   17/21	   66/93	   0.213*	  AT	   14/33	   3/21	   24/93	  TT	   1/33	   1/21	   3/93	  c.*111C>T	  	   CC	   17/31	   17/21	   62/89	   0.129*	  CT	   14/31	   3/21	   24/89	  TT	   0/31	   1/21	   3/89	  São	   Tomé	   and	   Príncipe	   is	   a	   small	   island	   in	  sub-­‐Saharan	  Africa,	  near	  equator,	  discovered	  by	  Portuguese	  navigators	  in	  the	  early	  1470s.	  	  It	  was	  a	   central	   warehouse	   for	   the	   assemblage	   and	  redistribu&on	  of	  slaves,	  first	  within	  the	  limits	  of	  the	   Gulf	   of	   Guinea,	   and	   later	   in	   the	  intercon&nental	   trade	   with	   the	   Americas,	  especially	   with	   Brazil4.	   	   The	   peopling	   of	   São	  Tomé	   might	   have	   provided	   one	   of	   the	   first	  examples	  of	  the	  combina&on	  of	  diverse	  African	  contribu&ons	   and	   European	   admixture	   that	  emerged	   from	   the	   overseas	   popula&on	  reloca&ons	   promoted	   by	   the	   Atlan&c	   slave	  trade.	  	  Table	  2.	  GJB2	  noncoding	  variants	  iden&fied	  in	  pa&ents	  and	  controls,	  and	  respec&ve	  genotypic	  frequencies.	  Table	  1.	  GJB2	  coding	  variants	   iden&fied	  in	  pa&ents	  and	  controls	  (all	   in	  simple	  heterozygosity),	  and	  respec&ve	  carrier	  frequencies.	  NA	  –	  Not	  Applicable;	  *Chi-­‐Square	  Test	  by	  Monte	  Carlo	  Simula&on	  1.	  Wonkam	  A,	  Bosch	  J,	  Noubiap	  JJN,	  Lebeko	  K,	  Makubalo	  N,	  Dandara	  C.	  No	  evidence	  for	  clinical	  u&lity	  in	  inves&ga&ng	  the	  connexin	  genes	  GJB2,	  GJB6	  and	  GJA1	  in	  non-­‐syndromic	  hearing	  loss	  in	  black	  Africans.	  South	  African	  Med	  J.	  2014;105(1):23.	  	  2.	  Bosch	  J,	  Lebeko	  K,	  Noubiap	  Nziale	  JJ,	  Dandara	  C,	  Makubalo	  N,	  Wonkam	  A.	  In	  Search	  of	  Gene&c	  Markers	  for	  Nonsyndromic	  Deafness	  in	  Africa:	  A	  Study	  in	  Cameroonians	  and	  Black	  South	  Africans	  with	  the	  GJB6	  and	  GJA1	  Candidate	  Genes.	  OMICS.	  2014;18(00):1–5.	  	  3.	  Javidnia	  H,	  Carson	  N,	  Awubwa	  M,	  Byaruhanga	  R,	  Mack	  D,	  Vaccani	  J-­‐P.	  Connexin	  gene	  muta&ons	  among	  ugandan	  pa&ents	  with	  nonsyndromic	  sensorineural	  hearing	  loss.	  Laryngoscope.	  2014;124(9):E373–6.	  	  4.	  Tomás	  G,	  Seco	  L,	  Seixas	  S,	  Faus&no	  P,	  Lavinha	  J,	  Rocha	  J.	  The	  peopling	  of	  São	  Tomé	  (Gulf	  of	  Guinea):	  origins	  of	  slave	  sedlers	  and	  admixture	  with	  the	  Portuguese.	  Hum	  Biol	  an	  Int	  Rec	  Res.	  2002;74(3):397–411.	  	  5.	  Matos	  TD,	  Simões-­‐Teixeira	  H,	  Caria	  H,	  Rosa	  H,	  O’Neill	  A,	  Fialho	  G.	  The	  controversial	  p.Arg127His	  muta&on	  in	  GJB2:	  report	  on	  three	  Portuguese	  hearing	  loss	  family	  cases.	  Genet	  Test	  Mol	  Biomarkers.	  2010;14(1):141–4.	  	  6.	  Del	  Cas&llo	  FJ,	  Rodríguez-­‐Ballesteros	  M,	  Alvarez	  A,	  Hutchin	  T,	  Leonardi	  E,	  de	  Oliveira	  C	  a,	  et	  al.	  A	  novel	  dele&on	  involving	  the	  connexin-­‐30	  gene,	  del(GJB6-­‐d13s1854),	  found	  in	  trans	  with	  muta&ons	  in	  the	  GJB2	  gene	  (connexin-­‐26)	  in	  subjects	  with	  DFNB1	  non-­‐syndromic	  hearing	  impairment.	  J	  Med	  Genet.	  2005	  Jul;42(7):588–94.	  	  

DFNB1 locus	analysis	in	São	Tomé	and	Príncipe	population

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DFNB1 locus analysis in São Tomé and Príncipe population

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